A single s.c. injection of haloperidol into ratson the morning of proestrus produced up to a 22-fold increase in serum prolactin and a significant increase in pituitary prolactin concentration. There was a significant decrease in serum LH and FSH levels and a complete block of spontaneous ovulation. Haloperidol significantly reduced hypothalamic PIF and LRF activities. The anti-ovulatory effect of haloperidol was overcome by the administration of exogenous LH. Haloperidol directly inhibited prolactin and LH release by the pituitary in vitro. These results suggest that the increased release and synthesis of pituitary prolactin by haloperidol is mediated by a decrease in hypothalamic PIF activity, and the reduction in release of LH by a decrease in hypothalamic LRF activity and perhaps also by a direct inhibitory action on the pituitary.