alpha-Melanocyte stimulating hormone (alpha-MSH) immunofluorescence was observed in the rat brain using a highly specific and well-characterized antibody. alpha-MSH was contained in the arcuate nucleus cell bodies and in varicose fibers distributed throughout the brain stem. alpha-MSH-containing fibers were present in various nuclei of the hypothalamus, preoptic area, septum, amygdala, mammillary body and central gray area. The distribution of alpha-MSH was verified by radioimmunoassay following microdissection of discrete brain nuclei. High concentrations of alpha-MSH were contained in the median eminence, medial preoptic, anterior hypothalamic, periventricular, paraventricular, arcuate, dorsomedial, posterior hypothalamic nuclei and bed nucleus of the stria terminalis. Moderate alpha-MSH concentrations were noted in the amygdala, septal area, central gray, dorsal raphe, and the nucleus tractus solitarius. Hypophysectomy did not significantly reduce the quantity of alpha-MSH fibers in the brain, thereby suggesting an extra-pituitary source of alpha-MSH. Lesions of the arcuate nucleus did, however, completely abolish alpha-MSH-like immunoreactivity. The alpha-MSH-like compound in the brain has immunochemical and electrophoretic properties similar to those of standard alpha-MSH. High pressure liquid chromatographic analysis demonstrated that the alpha-MSH immunoreactivity in the brain was comprised of one major component having a retention time identical with that of standard alpha-MSH, as well as 2 minor components. In male rats kept on a 12-h light-dark schedule (0600-1800 hours), there was a diurnal rhythm of alpha-MSH in the hypothalamic nuclei with peak content at 0900 in the arcuate and periventricular nuclei of the thalamus; at 1300 in the dorsomedial, paraventricular and anterior hypothalamic nuclei, and at 1700 in the medial preoptic nucleus. In the pineal gland a diurnal rhythm was also observed with a peak concentration at 1900. Six days of constant light, however, abolished the morning rise in alpha-MSH. Rats kept in constant dark for 6 days showed a marked increase in the alpha-MSH peak (12-fold) occurring at 0500. Normal diurnal rhythm of alpha-MSH was still observed in hypophysectomized rats. It is suggested that alpha-MSH may function as a neurotransmitter or as a neuromodulator in the brain. The extensive distribution of alpha-MSH in the brain suggests that it is involved in significant neuronal circuitry and supports the notion of a neuroregulatory role for this neuropeptide. This lays the groundwork for a rational approach to further study of possible interactions between alpha-MSH and other neuronal systems.