The binding of 125I-labeled rat prolactin (125I-rat PRL) to membranes from different regions of the rat brain was studied. Among these regions the hypothalamus showed the highest specific binding. Clearly detectable specific binding was also observed in substantia nigra, whereas it was very scanty in other brain regions. No significant sex differences in PRL binding to various brain regions were observed, except for hypothalamus where a higher binding was observed in female rats. The binding of 125I-rat PRL to hypothalamus from female rats was inhibited in a dose-dependent manner by both unlabeled rat and ovine PRL but not by several other polypeptide hormones. Scatchard analysis of the binding revealed the presence of the binding sites with low capacity and high affinity for rat ligand. Ovariectomy markedly decreased PRL binding in the hypothalamus; an even more pronounced decrease was found after hypophysectomy of female animals. A treatment with estradiol restored the PRL binding in the ovariectomized rats to above normal levels. These results of in vitro biochemical analysis together with the experimental modulation of hormonal status provide strong preliminary evidence for the presence of PRL binding sites in rat brain.