The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. alpha-MSH-like containing perikarya were localized in the infundibular region, mainly in the ventral hypothalamic nucleus. A rich plexus of immunoreactive fibers directed towards the ventral telencephalic region was detected. Reverse-phase high-performance liquid chromatography and radioimmunoassay were used to characterize alpha-MSH-like peptides in the frog brain. Chromatographic separation revealed that immunoreactive alpha-MSH coeluted with synthetic des-N alpha-acetyl alpha-MSH, authentic alpha-MSH and their sulfoxide derivatives. The heterogeneity of alpha-MSH-like material in the frog brain was in marked contrast with the figure observed in the intermediate lobe of the pituitary gland where only des-N alpha-acetyl alpha-MSH is present. These findings support the existence of discrete alpha-MSH immunoreactive neurons in the frog brain containing both desacetyl and authentic alpha-MSH.