The aim of the present study was to examine the acute and chronic effects of the gonadotropin-releasing hormone agonist (GnRH-a) leuprolide acetate (LA) on the expression of the steroidogenic acute regulatory protein (StAR), the cytochrome P450 side-chain cleavage enzyme (P450_scc), and steroid production in antral ovarian follicles obtained from prepubertal equine choriogonadotropin (eCG)-treated rats. Follicular contents of StAR and P450_scc proteins were measured by Western blotting following in vivo injection of eCG (control) and eCG LA (LA) to prepubertal rats. Treatment with eCG for 2 h resulted in no change in StAR protein content, but it was markedly increased at 4 and 8 h after hormone treatment. However, coadministration of eCG LA produced a significant increase (P < 0.05) in StAR protein levels at 2, 4, and 8 h when compared with eCG treatment. Acute and chronic treatment with either eCG or eCG LA did not alter the P450_scc protein levels in freshly isolated follicles. The increase in StAR protein expression following LA treatment was qualitatively similar to StAR mRNA expression, as determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Furthermore, administration of eCG demonstrated a time-dependent increase (2–8 h) in the levels of StAR mRNA, and these levels were markedly increased by eCG LA. However, the temporal response pattern of StAR mRNA was much greater at 2 h following LA administration when compared with controls. In addition, 48 h of LA treatment in eCG-treated rats resulted in a significant increase (P < 0.05) in follicular progesterone levels, whereas significant decreases in androgen (testosterone and androsterone) and estradiol levels were observed. Similar results were obtained when serum androgens and estradiol were measured, but serum progesterone levels were unchanged. Collectively, these findings demonstrate that the inhibitory effect of LA on ovarian androgen and estradiol levels is related to changes in the follicular levels of StAR protein and steroid production.