The role of serotonin (5-HT) in the regulation of steroidogenesis in the ovary via 5-HT2 receptor was studied in normal cycling rats and their preovulatory follicles. Oral administration of ketanserin (KET), a selective 5-HT2 antagonist, at 10 mg/kg BW at 1100h on the day of proestrus resulted in a marked decrease in serum estradiol levels immediately after the administration. 5-HT stimulated estradiol secretion by preovulatory follicles incubated for 5 hrs in Krebs-Ringer bicarbonate buffer. 5-HT (2.5 microM) -enhanced estradiol secretion was inhibited by KET (1-100 microM) in a dose-dependent manner. Secretion of progesterone and testosterone was also inhibited by about the same order of magnitude as estradiol. Addition of pregnenolone (1 microgram/ml) prevented the KET-inhibited progesterone production. Both testosterone and estradiol increased at the same ratio by the addition of progesterone (1 microgram/ml). The inhibition of secretion of these hormones was overcome by dbcAMP (0.1 mM). The present study suggests that 5-HT stimulates steroidogenesis in the metabolic pathway prior to pregnenolone in rat preovulatory follicles and the action of 5-HT could possibly be mediated by a 5-HT2 receptor.