Specific binding sites for 125iodine-prolactin are present in membrane particles obtained from the rat ventral prostate, human benign prostatic hyperplasia and prostatic adenocarcinoma. In the ventral prostate glands of young rats (1 to 4 months old) specific binding of 125iodine-prolactin is higher than in older animals (greater than 8 months old). Subcellular distribution studies revealed that specific 125iodine-prolactin binding activity is associated primarily with the 15,000 and 100,000 g particulate membrane fractions of the rat ventral prostate and human prostate glands. In rats between 2 and 4 months old significant increases in the prolactin binding activity in the 100,000 g membrane fraction of the ventral prostate are observed to occur without concomitant increases in prolactin binding activity in the 15,000 g fraction. The level of specific 125iodine-prolactin binding activity present in the human prostate gland is lower than that observed in the rat ventral prostate gland. Localization of specific prolactin binding sites in the rat ventral psotate and the human prostate gland suggests that prolactin could influence the function of these tissues directly.