We tested the effects of low concentrations of 17 beta-estradiol (E2) on the in vitro pituitary responsiveness to gonadotropin-releasing hormone (GnRH) in frogs (Rana pipiens). We first showed that the pH indicator phenosulfonpthalein (phenol red, PR) at levels normally used in the culture medium did not significantly affect pituitary basal or GnRH-stimulated gonadotropin secretion in male glands, as may occur in mammals. Incubation of male glands with E2 at 10 pg/ml for 24 hr in medium lacking PR did not alter basal follicle-stimulating hormone (FSH) or luteinizing hormone (LH) secretion, but significantly attenuated responsiveness of both gonadotropins to GnRH (0.2, 1, and 5 ng/ml); E2 did not affect responses at the highest dose of GnRH (25 ng/ml). GnRH responsiveness of female pituitaries was also inhibited by E2 at all doses of GnRH; 1 ng/ml E2 had greater effects than did 10 pg/ml. The present results demonstrate that E2 at concentrations commonly found in the circulation of both sexes have a consistent direct inhibitory effect on FSH and LH secretion at the level of the pituitary.