Seymour Kety had left the University of Pennsylvania in 1951 to become the first scientific director of the combined intramural research programs of the National Institute of Mental Health (NIMH) and the National Institute of Neurological Diseases and Blindness (NINDB). Sokoloff continued some of Kety's projects, and Kety continued to advise the research group, but without his leadership the group at Penn gradually withered. In 1953, Sokoloff, wishing to continue studying cerebral circulation and metabolism, joined the NIMH Laboratory of Neurochemistry (Cerebral Metabolism section) at Kety's invitation.

Before World War II, the National Institutes of Health (NIH) had been a rather modest research establishment with only one special institute, the National Cancer Institute. It began to grow rapidly in the post-war era. Institutes for research on heart disease and dental disease were established in 1948. The NIMH followed in 1949, with a broad mandate to support research into neurochemical processes underlying mental disorders, but also behavioral and social science research. The NINDB was established in 1950, to conduct and support research on the many neurological and sensory disorders (including cerebral palsy, epilepsy, multiple sclerosis, muscular dystrophy, cerebral vascular disease, and blinding diseases) that disabled or killed millions of Americans. Because research at both institutes focused on elucidating central nervous system physiology, the Surgeon General had designated the NIMH to administer the NINDB intramural research program along with its own. It was difficult at the time to separate basic research in neurological disease and mental illness. There were no scientifically validated theories regarding the origins of most neurological and psychiatric disorders, and (as Sokoloff had discovered) clinical research produced mostly descriptive results. There was no way to predict which basic research areas would yield valuable diagnostic or therapeutic information, and so having a joint program would avoid duplication of research. For these reasons, Kety, as Scientific Director, organized the NIMH/NINDB research program according to scientific discipline, rather than by disease category, and strongly encouraged multidisciplinary cooperation between the laboratories. Along with this, he allowed his scientists complete freedom to choose their research problems, as he believed this would generate more scientific discoveries and attract more young scientists to the program.

Sokoloff thrived in this supportive, interdisciplinary environment, where scientists shared their expertise and a young investigator could sharpen his skills. He continued using the N2O method to study the effects of normal aging and dementia on CBF and energy metabolism in humans. As was the case at the University of Pennsylvania, his research group found that measuring blood flow and metabolism with N2O was useful for tracking overall changes in the brain, but could not detect effects from mental exercise (for example, solving arithmetic problems), schizophrenia, or alcohol and drug intoxication. They reasoned that whatever metabolic increases resulted from these functional activities must happen in specific local areas of the brain, and be too small to register in the overall blood flow measurement. To look at local blood flow volume as an indicator of metabolic activity in various parts of a conscious brain, they developed a chemically inert radioactive iodine tracer, [131 I]trifluoroiodomethane. (A tracer is a compound in which one of the constituent elements has been replaced with a radioactive isotope of that element--in this case, the iodine. The radioactive isotope is often called a "label" or a "tag". Radioactive isotopes have a different number of neutrons in their nuclei than the common form of the element, giving them a different atomic mass, and making them unstable. They begin to decompose to more stable elemental forms, and give off radiation in the process. But in chemical reactions, they will behave just like the common form of the element, and their radioactivity makes it possible to follow the path of the labeled compound with a radiation detector. Some radioactive isotopes occur naturally, e.g. carbon 14 (14C), but they can also be made artificially by bombarding the desired element with nuclear particles in a cyclotron. Radioactive isotopes decay at different rates; some disintegrate so quickly that they can't be used for experiments, while others, like uranium 235, stay radioactive for billions of years.) Sokoloff's team also constructed an equation, similar to the one used for the N2O experiments, that described the concentration of tracer in the tissue at a given time after administration of the tracer into the blood, the total time course of the tracer concentration in the arterial blood between administration of the tracer and measurement of the tissue concentration, the time after tracer administration, and the relative solubility of the tracer between the tissue of interest and the blood. In their experiments, a solution of the tracer was infused into a living cat's vein for about one minute, during which time they continuously sampled the arterial blood to determine the time course of the tracer concentration in the blood. To measure the concentrations of the tracer in particular parts of the brain at a particular moment, the animal was humanely killed and its head frozen in liquid nitrogen. The frozen brain was cut into thin cross-sections, and the sections placed on x-ray film. When the film was developed, the image (called an autoradiogram) showed the distribution of the radioactive tracer in the structures of the brain. The darker the film, the greater the concentration of the tracer in the tissue. The investigators could then use a densitometer (an instrument that measures the degree of darkness in film) to relate the darkness in a given region of the film image to the concentration of the tracer in the tissues represented. The autoradiograms showed the anatomical components of the brain quite clearly, and demonstrated significant differences in blood flow rates to them. Because the concentrations of tracer in the various tissues were closely related to the rates of blood flow to them, the autoradiograms were essentially pictures of the relative rates of blood flow in those tissues at a given moment. The group's 1955 report, which described images obtained from the brains of cats with eyes open or closed or stimulated with light flashes, was the first published demonstration of functional brain imaging. They later used antipyrine and iodoantipyrine labeled with 14C for their experiments, as those tracers were easier to make.

Mapping local cerebral blood flow with this radioactive tracer technique was a promising start in understanding brain function. Blood flow, however, could provide only an approximate measure of activity. What Sokoloff wanted was a more direct way to track the brain's energy metabolism, i.e., its use of oxygen and glucose. Using oxygen as a label was ruled out because the radioactive isotope of oxygen has a half-life too brief to use with radiography, and labeled products of oxygen consumption (carbon dioxide and water) are removed from the brain too quickly by the blood flow to be accurately measured. Measuring glucose consumption was a more promising approach because (unlike other body tissues) the brain normally uses only glucose to produce energy. Thus, glucose consumption would be a very good measure of energy metabolism. For several years, Sokoloff and his colleagues tried to develop a way to use glucose labeled with the radioactive isotope 14C. However, they found that, again, the labeled end products didn't stay in the tissue or blood plasma long enough to be accurately measured. Sokoloff put this work aside to pursue another project.

Sokoloff's primary project during the 1950s and 1960s was the study of thyroid hormones and protein synthesis, inspired by the inconclusive experiments he did as a research fellow at the University of Pennsylvania. Those studies showed that treating hyperthyroidism had no effect on measures of brain energy metabolism, so the question remained: If thyroxine didn't affect oxygen consumption in the brain, then what role did it have there? He had earlier learned of the intimate role played by thyroid hormone in the regulation of protein synthesis in most tissues, and he set out to examine the mechanism of this effect. To do this, he spent several years expanding his knowledge and skills in biochemistry, mentored by his NIH colleague Seymour Kaufman. In collaboration with Kaufman, he set up a method to analyze protein synthesis in vitro, and after many years of repeated long experiments, they were able to demonstrate that thyroxine stimulates amino acid incorporation into protein both in vitro and in vivo. His earlier experiments had failed to detect an increase in cerebral oxygen consumption in hyperthyroidism because the amount of oxygen consumed by thyroxine-stimulated protein synthesis in the mature brain is quite small compared with that required for the brain's normal energy metabolism (i.e., breakdown of glucose.) This research experience was a turning point for Sokoloff. The capability that biochemistry seemed to offer for definitive solutions pulled him away from physiology and he became "oriented toward relationships between biochemical processes and physiological functions, particularly in the nervous system." He continued the thyroxine and protein synthesis work for another decade, adding much to scientific understanding of the role of thyroxine in brain maturation.