Serotonin plays a hormonal function in several nonneuronal peripheral tissues, such as the ovaries. Our aim was to investigate whether there is a modulatory action of serotonin on gonadotropin-induced steroid secretion of human granulosa cells. In granulosa cell culture, serotonin was administered alone or in combination with luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Also, granulosa cells were transferred into a dynamic superfusion apparatus and challenged by FSH and LH alone or along with serotonin. Estradiol and progesterone concentrations of samples were measured by radioimmunoassay. As expected, administration of FSH, LH, and serotonin alone resulted in a significant estradiol and progesterone release in cell culture, as well as a significant increase in progesterone release in dynamic superfusion system. In cell culture, co-administration of serotonin with gonadotropins had no additive effect on gonadotropin-induced secretion of progesterone, while it further augmented that of estradiol. In superfusion system, when gonadotropins were added along with serotonin, the increase in progesterone release was markedly less, while peaks of hormone response were remarkably prolonged compared to challenges by LH and FSH alone. The observed effects of serotonin on gonadotropin-induced steroid release of granulosa cells may reveal further details about the regulation of granulosa cell function.