Serotonin (5-hydroxytryptamine, 5-HT), considered to be a synaptic transmitter within the central nervous system, has been shown to be localized mainly in the paleocortex of the brain. Others have shown disturbances of normal 5-HT metabolism to be associated with mental illness and disease. Attempts to inject tritium labelled 5-HT into cerebrospinal fluid of the rat brain for anatomical localization studies were·made. The following observations were noted, (1) there exists a perivascular space in which fluid flows and distribution is relatively uninhibited as compared to subarachnoid space, (2) the labelled 5-HT penetration of brain tissue parenchyma is restricted and radioactivity is attenuated going away from the surface of access, and (3) the injected labelled 5-HT is absorbed into areas believed to contain true serotonergic fibers, especially throughout the paleocortex. Suggestions concerning the anatomical importance of the perivascular space observed are made. Also comparisons of 5-HT brain localization with metabolism and physiology of 5-HT are made in relation to mental illness.