Our previous in vivo and in vitro studies revealed that prolactin (PRL) affected luteal function during the first days of the porcine estrous cycle. Since the mechanism by which the luteotrophic action of PRL might be mediated was not elucidated, the goal of the present study is to investigate the effects of short term, in vivo administration of PRL on in vitro functions of hypothalamic explants, adenohypophyseal cells and luteal cells of sows. Injections of PRL or saline (performed every 2h) started shortly after the preovulatory LH surge and lasted for 2 or 3 days. Peripheral blood plasma for determination of LH, PRL and progesterone (P(4)) was sampled at 4h intervals. Ovaries, pituitaries and the stalk median eminence (SME) dissected after slaughter were used for in vitro studies. Luteal and adenohypophysial cells as well as hypothalamic tissue were incubated/cultured with different treatments. Medium and plasma levels of GnRH, LH and P(4) were quantified by radioimmunoassays (RIAs). Corpora lutea (CL) were used for LH/human chorionic gonadotrophin (hCG) receptor analysis. In vivo and in vitro treatment with PRL increased the in vitro GnRH release by hypothalamic explants (P<0.05). GnRH-stimulated LH production was enhanced in PRL-treated sows compared to that of control sows (P<0.05). PRL injections had no effect on plasma P(4) concentrations during the treatment period. However, luteal secretion of P(4) (P=0.06) and LH/hCG receptor concentration (P=0.079) tended to be higher in PRL-treated sows in comparison to those of controls. The results indicate that PRL may be involved in the regulation of the hypothalamic-pituitary-ovarian axis at the beginning of the luteal phase of the porcine estrous cycle.