Immunoreactive (IR) prolactin was localized immunocytochemically in cell bodies in the mediobasal hypothalamus and in fibers in many regions of the rat brain. The cell bodies were found in the arcuate nuclei and the adjacent areas ventral to the ventromedial nuclei. Fiber projections extended rostrally to and/or through the anterior hypothalamus, preoptic area, nucleus accumbens, septum, diagonal bands of Broca, caudate-putamen, frontal cortex and accessory olfactory bulb; laterally to the amygdala, especially the central nucleus and some parts of the medial nucleus; caudally to and/or through the midbrain central gray, reticular formation, parabrachial region, and several portions of the lower brain stem and spinal cord extending to sacral levels. The system appears to be essentially identical to that containing pro-opiomelanocortin (POMC) and its processed peptides, as shown by double immunocytochemistry. Preabsorption of the antiprolactin antiserum with either prolactin or the 16,000-dalton N-terminus of POMC eliminated immunoreactivity in the brain. Preabsorption with other POMC-derived peptides, including β-lipotropic hormone, β-endorphin, met-enkephalin, adrenocorticotrophic hormone (1–24), corticotropin-like intermediate lobe peptide, α- and γ-melanocyte-stimulating hormones and an octapeptide region of the N-terminus of POMC bearing some homology with prolactin, did not eliminate immunoreactivity in the brain. Similarly, preabsorption with growth hormone, luteinizing hormone, follicle-stimulating hormone, motilin or fetuin did not eliminate immunoreactivity in the brain. The antiprolactin antiserum also recognized all cells in the intermediate lobe and a subset of cells in the anterior lobe of the Snell dwarf mouse pituitary. This immunoreactivity was eliminated by preabsorption of the antiserum with prolactin or with the 16,000-dalton N-terminus of POMC. These results suggest that IR prolactin in the brain may be related to the N-terminus of POMC. Additional results based on one- and two-dimensional gel electrophoresis and immunoblotting indicate that the antiprolactin antiserum used in the majority of the immunocytochemical studies recognized a number of proteins.