We have previously shown that the proliferative index (PI), as determined by flow cytometry of luteinized granulosa cells obtained at oocyte retrieval, is greater in ovulation induction regimens which include the GnRH analog (GnRH-a) leuprolide acetate than those using human menopausal gonadotropin (hMG) only. Specific growth factors or intrafollicular hormones may contribute to this leuprolide acetate-induced difference in cell cycle kinetics. We examined whether differences in the PI of these granulosa cells are associated with the alterations of follicular fluid content of Mullerian-inhibiting substance (MIS) and other intrafollicular hormones including FSH, estradiol, progesterone, androstenedione, and testosterone. The control group consisted of follicular fluid obtained from 18 follicles from 4 women receiving hMG alone. The GnRH-a treated group consisted of follicular fluids obtained from 55 follicles aspirated from 18 women receiving GnRH-a in addition to hMG. One-way analysis of variance using log-transformed data and expressed as geometric means with 95% confidence intervals, demonstrated that the follicles from the control group had a significant 14-fold higher concentration of 2.46 ng/mL MIS, 95% CI (1.8-4.8) vs. 0.18 ng/mL, 95% CI (0.13-0.24) P < 0.0005, a 3-fold higher concentration of 17.55 nmol/L androstenedione, 95% CI (14.6-20.9) vs. 5.76 nmol/L, 95% CI (3.1-10.5) P < 0.02, and a 1.5-fold higher concentration of 29.43 nmol/L testosterone 95% CI (22.5-38.14) vs. 19.3 nmol/L, 95% of CI (11.1-33.9) P < 0.01 than GnRH-a treated follicles, although the PI value in controls was half that of the GnRH-a group. These data demonstrate that GnRH-a induced differences in granulosa cell cycle kinetics are associated with alterations of MIS and androgen intrafollicular fluid content and suggest that MIS may be a mitotic inhibitor of human granulosa cells.