The present study examines prolactin PRL-like immunoreactivity (PRL-LIR) in the rat central nervous system and describes the distribution of labeled perikarya and fibers using a specific antiserum to ovine PRL. This antiserum does not cross-react with molecules of the pro-opiomelanocortin (POMC) family and recognizes rat PRL. PRL-LIR cell bodies are found exclusively in the lateral hypothalamic area surrounding the fornix, especially dorsolateral to it. No labeled cells are detectable in any other part of the brain, including the arcuate nucleus. Labeled fibers are dispersed in almost all parts of the brain. Dense plexuses are observed in the hypothalamus, midline thalamus nuclei, bed nucleus of stria terminalis, raphe dorsalis, and locus coeruleus. There is no apparent decrease in the number of PRL-LIR cell bodies and fibers in hypoprolactinemic mutant rats or after hypophysectomy, suggesting that central PRL is synthesized in such hypothalamic neurons. Comparison of PRL and alpha-melanocyte-stimulating hormone immunostainings provides evidence that the PRL network is independent of those of POMC and melanin-concentrating hormone. The present results support the hypothesis of two independent PRL systems: one peripheral (pituitary gland) and the other cerebral. Concerning the functional role of brain PRL, its widespread projections suggest that PRL is involved in multiple regulations. The presence of PRL-LIR in brain areas involved in sleep-wake control is a strong argument for its role in such a regulation.