Abstract Hyperprolactinemia suppresses luteinizing hormone (LH) and prolactin (PRL) secretion under a variety of experimental conditions. The secretion of both of these hormones is regulated at the hypothalamic level by several neurotransmitters, including serotonin (5-HT). Therefore, we examined the effect of hyperprolactinemia on 5-HT neuronal activity in key hypothalamic areas that are rich in 5-HT terminals and which are known to regulate the release of LH and PRL. Young cycling virgin rats were ovariectomized (day 0). From days 11–16, animals were injected with ovine prolactin (oPRL, 4mg/kg, s.c.) or vehicle every 8 h. On day 14, one-half of the oPRL- and vehicle-treated rats were implanted with 20-mm long Silastic capsules containing estradiol (180 μg/ml). On day 16, animals were killed at 08.00, 12.00 or 18.00 h or treated with pargyline (75 mg/kg) and killed 10 min later. Trunk blood was collected and serum was radioimmunoassayed for LH and endogenous rat PRL (rPRL). Brains were removed, frozen, sectioned and the medial preoptic, suprachiasmatic, and arcuate nuclei, median eminence and globus pallidus were microdissected. Serotonin was measured using high pressure liquid chromatographic methodology. We were unable to detect any feedback effect of hyperprolactinemia on 5-HT turnover in any brain area of ovariectomized or ovariectomized estradiol-treated rats at any time of day that we examined. Several potential reasons for the absence of an effect of hyperprolactinemia on serotonergic function are discussed.