Objective: To examine the relationship of serum mullerian-inhibiting substance (MIS), E-2, free-T, LH, and FSH in untreated women with polycystic ovary syndrome (PCOS) and in women with normal menstrual cycles. Design: A prospective study. Setting: University Departments of Obstetrics and Gynecology and Surgery. Patient(s): Twenty-seven women with PCOS and 20 women with normal menstrual cycles. Intervention(s): Serum was collected from women with PCOS and from normal women during the early follicular phase of the menstrual cycle, stored frozen until assayed. Main Outcome Measure(s): Serum levels of MIS, E-2, free-T, TSH. LH, and FSH were measured. Result(s): Serum mullerian-inhibiting substance levels in PCOS patients were significantly higher compared with normal women (+/- SE; 5.3 +/- 0.7 and 1.4 +/- 0.2 ng/mL, respectively). An inverse correlation (r = -0.5965) was found between serum levels of MIS and E-2 in PCOS women, but not in normal women. Women with PCOS had higher serum LH levels than those of normal women (15.2 +/- 1.2 and 5.0 +/- 0.7 mIU/mL). Conclusion: In this study, women with PCOS have significantly higher serum MIS levels than normal women. The inverse relationship between mullerian-inhibiting substance and E-2 levels suggests that MIS may modulate ovarian E-2 synthesis and have a role in the disordered folliculogenesis characteristic of PCOS. (Fertil Steril(R) 2002;77:141-6. (C) 2002 by American Society for Reproductive Medicine).