An immunocytochemical localization of alpha-melanocyte-stimulating hormone (alpha-MSH) as well as ACTH and a fragment (16K) of the common precursor of ACTH and beta-lipotropin (beta-LPH) was performed in rat brain. Two different groups of neuronal cell bodies showing alpha-MSH-like immunoreactivity (alpha-MSH-LI) were observed in the hypothalamus. One group of neurons located in the arcuate nucleus was shown to contain not only alpha-MSH-LI, but also ACTH and the 16K fragment. A second category of alpha-MSH-LI-containing neurons was characterized by the complete absence of staining for ACTH and 16K fragment. These neurons were mainly located in the dorsal-lateral portion of the hypothalamus. Immunoelectron microscopy showed that immunostaining for alpha-MSH was restricted to dense core vesicles in the positive perikarya. Nerve fibers staining for alpha-MSH (but not for ACTH and 16K fragment) were also observed outside the ACTH-beta-LPH pathway, especially in the cortex, caudate-putamen nucleus and hippocampus. These findings strongly suggest the presence of two different neuronal systems reacting with antibodies to alpha-MSH.