Immunohistochemical studies have identified immunoreactive prolactin (IR-PRL) in the hypothalamus and other areas of the rat brain. However, immunocytochemical techniques make it difficult to quantify the amount of antigen localized in a specific region. In this study, IR-PRL was extracted from selected regions of the rat brain, consisting of the median eminence, dorsal and ventral hypothalamus, thalamus, amygdalae, cerebellum, cortex, hippocampus, septum, pons-medulla, and olfactory lobes, and the concentrations of IR-PRL were determined by radioimmunoassay. Whereas IR-PRL was detected in all brain regions in both the male and the female rat brain, the concentrations of IR-PRL in the female rat were significantly greater than those measured in the corresponding region of the male rat brain. In the female rat, hypophysectomy significantly reduced, but did not eliminate, the concentration of IR-PRL in hypothalamus, amygdala, thalamus, and pons-medulla. In contrast, hypophysectomy did not affect the concentration of IR-PRL in any brain regions of the male rat. Injection of colchicine into the lateral ventricle decreased the concentration of IR-PRL in the median eminence and increased the concentration of IR-PRL in the ventral hypothalamus in male and female rats. In addition, extracted hypothalamic and pituitary IR-PRL displayed similar dilution curves in the PRL assay and elution patterns on Sephadex G-100. These data indicate that both the male and the female rat brain contains an IR-PRL-like material with physicochemical properties similar to those of pituitary PRL. This material is differentially distributed in the male and female brain and is found in greater concentrations in the female brain. Finally, the maintenance of IR-PRL in the brain in hypophysectomized rats and the decrease in median eminence IR-PRL in colchicine-treated rats suggest that this IR-PRL-like material is synthesized in the central nervous system.