Despite the probable inhibitory effects of GnRH analogues on ovarian steroidogenesis in vitro, their association with assisted reproduction protocols shows favorable results. This suggests that there are important differences in the behaviors of these drugs when administered in vivo versus in vitro. To clarify these differences, this study was designed to analyze the effect of leuprolide acetate (LA) on ovarian steroidogenesis in women undergoing In Vitro Fertilization (IVF). A prospective, randomized open label study was conducted on 14 women (26–35 years): seven receiving only gonadotrophins (Group 1) and seven receiving gonadotrophin plus LA at 1mg/day (Group 2). The LA in vivo effect was determined with serum and follicular fluid (FF) samples and via luteinized granulosa cell cultivation (GCC), where cells were obtained during oocyte retrieval after ovarian hyperstimulation. In vitro analysis was performed via addition of LA to GCC only for Group 1 (without LA) at progressively higher concentrations (0, 10−12, 10−9 and 10−6 M). In vivo, the main observation was a reduction in androgen production in Group 2, represented by lower androstenedione production in FF (G1 = 6479 ± 3458; G2 = 3021 ± 1119 ng/ml; p = 0.04) and a lower testosterone peak in GC at 96 h (G1 = 0.64 ± 0.12 ng/ml; G2 = 0.50 ± 0.19ng/ml; P = 0.02), but a higher fertilization rate (G1 = 67%; G2 = 83%; p = 0.009). In vitro, testosterone, estradiol and progesterone were also reduced by LA, even though this reduction occurred for progesterone only at the highest LA dosage (10−6 M; 606.0 ± 114.3 ng/ml versus 1524.0 ± 246.5 ng/ml; p = 0.02). Results show that LA reduces ovarian steroidogenesis in vivo by essentially inhibiting androgen synthesis; whereas, in vitro, ovarian steroidogenesis is reduced overall.