The distribution and concentration of alpha-MSH in the rodent brain has been determined by radioimmunoassay. The limbic system contained substantial quantities of alpha-MSH. Forty per cent of the alpha-MSH present in the brain was localized in the hypothalamus, with the highest concentration of alpha-MSH in the arcuate nucleus. More than 40% of the extrahypothalamic alpha-MSH in the brain was found in the following areas: midbrain (16%), preoptic area (13%), septum (7%), and thalamus (7%). To determine the source of the hypothalamic and extrahypothalamic alpha-MSH, the anterior hypothalamic preoptic area of the brain was surgically separated from more caudal diencephalic structures, and the arcuate region of the hypothalamus was surgically isolated from the remainder of the brain. Following these deafferentations, no significant reduction in hypothalamic alpha-MSH levels was observed; however, a significant reduction in extrahypothalamic alpha-MSH level was demonstrated. This dramatic decrease of alpha-MSH in extrahypothalamic areas of the rodent brain strongly suggests that the bulk of the extrahypothalamic alpha-MSH arises from neuronal perikarya in the arcuate region. These findings are consistent with the hypothesis that a population of neuronal cell bodies producing alpha-MSH originate in the arcuate region of the hypothalamus and that they send axonal projections to many areas of the limbic system and brain stem.