Our objective was to study the direct action of a GnRH-I agonist, leuprolide acetate (LA), on ovarian steroidogenesis in preovulatory follicles obtained from equine chorionic gonadotropin (eCG)-treated rats. Previously, we have demonstrated an inhibitory effect of LA on steroidogenesis and follicular development. In this study, we tested the hypothesis that gonadotropin-releasing hormone (GnRH) exerts its negative effect on follicular development by inhibiting thecal cytochrome P-450 C17 (P450C17) alpha-hydroxylase expression and, consequently, androgen synthesis. Studies were carried out in prepubertal female rats injected with either eCG (control) or eCG plus LA (LA) and killed at different time points. Immunohistochemical studies indicated that LA induced steroidogenic acute regulatory protein (StAR) expression mainly in theca cells of preantral and antral follicles. In addition, serum progesterone levels increased significantly (P < 0.05), whereas those of androsterone decreased (P < 0.05) after 8 h of LA treatment. This inhibition caused by LA seemed to be a consequence of the decreased expression of follicular P450C17 alpha-hydroxylase, as demonstrated by Western blot and RT-PCR techniques. In vitro studies using follicles isolated from 48-h-eCG-treated rats and cultured with LA showed a significant (P < 0.05) inhibition of FSH-induced androsterone follicular content as well as P450C17 alpha-hydroxylase protein levels, as determined by Western analysis. However, LA increased StAR protein expression in these follicles without significant changes in P450scc enzyme levels. Taking all these findings into account, we suggest that GnRH-I exerts a direct inhibitory action on gonadotropin-induced follicular development by decreasing the temporal expression of the P450C17 enzyme and, consequently, androgen production, thus reducing the supply of estrogens available to developing follicles.