Prolactin (PRL) effects on the female reproductive system have been presumed to occur primarily at the hypothalamic-pituitary level. The following studies were designed to evaluate whether PRL can directly alter gonadotropin actions at the ovarian level. In the first experiment, 10 groups of 7 cycling adult female rats were given a daily dose of pregnant mare's serum (PMS: 25 IU) and either saline (SAL) or PRL (0.25, 0.8, 2.5, 8, or 25 micrograms) twice daily for 4 days. In the second experiment, PMS (6 doses: 0-75 IU/d; 16 animals/dose) was administered to all animals while half the animals at each PMS dose received PRL (25 micrograms twice daily) and half received an equal volume of diluent. Finally, hypophysectomized (hypox) adult rats (n = 5-6/group) received 25 IU PMS/d and PRL (0-75 micrograms) twice daily. An additional group received 0 PRL and 0 PMS. Ovarian weight and histology were evaluated at the completion of each study. In the first experiment, PRL inhibited PMS-stimulated ovarian weight gain in a dose-dependent manner (p less than 0.01). Numbers of preantral (p less than 0.005) and antral (p less than 0.05) follicles were decreased in animals receiving an inhibitory dose of PRL (25 micrograms BID) compared to controls. In the second experiment PRL (25 micrograms BID) again inhibited PMS-stimulated ovarian weight (p less than 0.01) at all doses of PMS. Finally, in hypox animals, PRL inhibited PMS-stimulated ovarian weight gain (25 and 75 micrograms PRL: p = 0.001), and mean number (p less than 0.001) and diameter (p less than 0.001) of antral ovarian follicles (8-75 micrograms PRL) compared to controls. In summary, administration of PRL inhibited PMS-stimulated ovarian weight gain, and antral follicle diameter (in hypox animals only) and number in adult female rats suggesting that in states of hyperprolactinemia, PRL alters gonadotropin-mediated activities (i.e., folliculogenesis) directly at the ovarian level in addition to its hypothalamic and pituitary actions.