Recent immunocytochemical studies have demonstrated the existence of two different neuronal systems containing alpha-MSH-like material in the brain: one originating from the arcuate nucleus and the other one from the dorsolateral hypothalamus. The aim of the present study was to further characterize alpha-MSH in these two discrete regions of the rat diencephalon. Intracerebroventricular administration of colchicine resulted in a marked decrease in the number of ACTH and beta-endorphin nerve fibers and a significant reduction in ACTH and beta-endorphin content in the dorsolateral hypothalamus. Conversely, colchicine treatment did not alter alpha-MSH, ACTH or beta-endorphin content in the arcuate nucleus and did not significantly affect alpha-MSH concentration in the dorsal region. Reverse-phase high-performance liquid chromatography showed that the major alpha-MSH-like compound localized in the dorsal hypothalamus co-migrated exactly with synthetic alpha-MSH, whereas the arcuate nucleus contained 5 peptides cross-reacting with alpha-MSH antibodies, 4 of them being different from standard alpha-MSH. Significant amounts of biologically active melanotropin, which migrated on Sephadex G-25 columns like synthetic alpha-MSH, were also detected in both the arcuate nucleus and dorsolateral hypothalamus. Taken together, these data demonstrate that the alpha-MSH cell bodies located in the dorsolateral hypothalamus specifically produce authentic alpha-MSH, whereas the alpha-MSH cell bodies in the arcuate nucleus also contain ACTH, beta-endorphin and several peptides immunologically related but not identical to alpha-MSH.