The effects of GnRH and a potent GnRH analogue (D-Ala6-des-gly-NH2-GnRH-ethylamide) on steroidogenesis in isolated preovulatory follicles of PMSG-treated immature rats were examined in short-term incubations and compared to the effect of LH. Steroids were analyzed by RIA. GnRH stimulated the accumulation of pregnenolone (2-fold), progesterone (4-fold), 20 alpha-OH-progesterone (38-fold), androstenedione (4-fold), testosterone (3-fold), and oestradiol-17 beta (2-fold) during a 6 h incubation. The time-course of stimulation was the same for each of the steroids analyzed, with a significant effect at 4 and 6 h, but not at 2 h of incubation. Dose-response curves were similar for each steroid, and the GnRH analogue and GnRH gave parallel curves with minimal effective concentrations being 1 and 10 ng/ml, respectively. The stimulatory effect of LH was more pronounced and rapid (2 h) than that of GnRH. During prolonged incubation (6-8 h) with GnRH or LH there was evidence for an inhibition of testosterone production, probably due to suppression of the C21-side chain cleavage enzyme. Thus, the qualitative response to GnRH on follicular steroidogenesis resembled that of LH in some but not all respects. The differences in time-course and maximal steroid secretion between GnRH and LH are compatible with different mechanisms of action in the follicle.