The regulation of human prolactin (PRL) secretion by gonadotropin-releasing hormone (GnRH) was evaluated with human pituitary monolayer cell cultures. Synthetic GnRH stimulated PRL secretion when exposed to cells in an estrogen-depleted environment. This response was inhibited by pretreatment of the cells with 17β-estradiol (E2). A 10-5 M GnRH antagonist inhibited luteinizing hormone (LH) but not PRL secretion when cells were maintained in an estrogen-depleted environment. However, the GnRH antagonist inhibited basal PRL secretion when cells were maintained in medium containing 10-8 M E2. The 10-5 M GnRH antagonist, when coincubated with 10-5 M GnRH inhibited the release of PRL in an estrogen-depleted environment. However, coincubation of the 10-5 M GnRH antagonist with 10-5 M thyrotropin-releasing hormone (TRH) failed to inhibit PRL secretion. Incubation of 10-8 M TRH and 10-8 M GnRH produced a synergistic release of PRL in an estrogen-depleted environment. These observations led us to conclude that GnRH stimulates PRL secretion by direct action on human pituitary cells and that GnRH acts either via the gonadotrope or through receptors on the galactotrope other than that acted upon by TRH to release PRL.