Hyperprolactinemia adversely affects reproductive functions, presumably through an effect at the hypothalamic level. Given the numerous published reports linking hypothalamic serotonin (5-hydroxytryptamine, 5HT) mechanisms to the regulation of gonadotrophin secretion, we sought to determine the effects of experimentally induced hyperprolactinemia on ovarian steroid-induced increases in serum LH levels and region-specific hypothalamic 5HT synthesis in ovariectomized rats. In the first study, bilaterally ovariectomized Sprague-Dawley rats either received two pituitary homo-grafts implanted beneath the left kidney capsule or were sham-grafted. Both groups of rats were injected subcutaneously with 5 µg/100 g of estradiol benzoate (E2) in corn oil vehicle at 08.00 h, 1 and 2 days before serum collection and 5 mg/100 g of progesterone (P) in corn oil vehicle at 07.00 h on the day of serum collection. Blood samples were collected via chronic indwelling jugular cannulae from each rat at 10.00, 12.00, 13.00, 14.00, 16.00 and 18.00 h. A statistically significant elevation in serum LH levels was detected at 13.00, 14.00 and 16.00 h. This increase in serum LH levels was significantly attenuated in rats bearing pituitary homografts, an effect attributed to the high serum PRL levels measured in these animals. In the second study, bilaterally ovariectomized Sprague-Dawley rats were divided into three experimental groups: (1) rats bearing two pituitary homografts and injected with E2 and P on the schedule and at the dosages previously described, (2) sham-grafted rats injected with E2 and P on the schedule and at dosages previously described and (3) sham-grafted rats injected with corn oil vehicle only. The rats were then injected intravenously with 10 mg/100 g of NSD-1015 6 h after P injection and 30 min prior to killing the rats. Accumulation of 5-hydroxytryptophan in various hypothalamic areas was then assayed by liquid chromatography with electrochemical detection as a relative index for the level of 5HT synthesis. Administration of these ovarian steroids produced an increase in estimated 5HT synthesis in the preoptic area-anterior hypothalamus (POA-AH) but not in the median eminence or mediobasal hypothalamus. The E2 + P-induced increase in 5HT synthesis in the POA-AH was significantly blunted in rats with pituitary homografts. Considering the postulated role a 5HT mechanism within the POA-AH plays to facilitate ovarian steroid-mediated surges in LH secretion, hyperprolactinemia may inhibit LH secretion through its attenuation of ovarian steroid stimulation of 5HT synthesis in the POA-AH