The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH) was studied in the rat brain with an immunoperoxidase technique. alpha-MSH-containing cells were found in the arcuate nucleus of the hypothalamus. Cells staining for alpha-MSH were also localized in the intermediate lobe of the pituitary. alpha-MSH-containing nerve fibers extended throughout regions of the hypothalamus, thalamus, and midbrain. Two weeks after hypophysectomy, alpha-MSH-positive cells anf fibers were still present in the brain. These results indicate that alpha-MSH of non-pituitary origin is synthesized and stored by neural structures in the rat brain. The detection of alpha-MSH by radioimmunoassay in the rat brain and pituitary supports these observations.