A role of prolactin (PRL) in ovarian function has been suggested in several species, but not unequivocally established except in the rat. We, therefore, examined the presence of specific receptor for PRL in ovaries of rat, cow, and human. Human PRL (hPRL) labelled with 125I by the lactoperoxidase method was shown to be capable of specific binding to rat mammary tissue homogenate. Human, cow, and rat ovarian homogenates and/or partially purified plasma membranes were also shown to specifically bind 125I-hPRL. Binding was a saturable phenomenon and was dependent on receptor protein concentration. Optimal binding was observed at pH 7.0 and at 37°C. Binding was reversibly inhibited by exposure of membranes to pH 10.0 and irreversibly destroyed by exposure to pH 3.0. Bound 125I-hPRL was displaceable by unlabelled human, ovine, and bovine PRL but not by FSH or LH. However, human chorionic somatomammotrophin (hCS) and hGH showed some competition with 125I-hPRL. Number of binding sites/mg protein was lowest (0.8 × 10−12 m) during metoestrus and increased during dioestrus (11 × 10−12 m) reaching the maximum number at pro-oestrus (24.6 × 10−12 m). These results demonstrate that presence of specific PRL receptor in the ovaries and are consistent with a role of PRL at the ovarian level.