The concentration of melanocyte-stimulating hormone (melanotropin; MSH) within the septal region of the brain increases during the fever, and septal injections of MSH are antipyretic. Corticotropin-releasing factor (CRF), when injected intracerebroventricularly, is also antipyretic. Using sensitive radioimmunoassays of microdissected tissue extracts, we established the presence of immunoreactive MSH (IRMSH) and CRF (IRCRF) within discrete central nervous system sites of the rabbit. Leukocytic pyrogen-induced fever and hyperthermia due to heat exposure did not alter concentrations of IRMSH or IRCRF in tissue extracted from preoptic-anterior hypothalamic or midbrain central gray regions. However, significantly greater levels of IRMSH were detected in septal extracts of febrile rabbits than in similar extracts from afebrile controls or heat-stressed animals. A significant decrease in IRCRF was detected in paraventricular nucleus extracts from febrile animals compared with extracts from afebrile controls or heat-stressed rabbits. Our results support the hypothesis that these central peptides have a role in temperature control during fever. Since no changes were detected in extracts from hyperthermic rabbits, it appears that changes in concentration of these neuropeptides within particular brain regions are specific to the febrile state and are not caused by elevation of body temperature or by nonspecific stress.