We investigated the mechanism of cyclophosphamide (CTX) induced ovarian toxicity by studying the effect of an activated form, 4-hydroperoxycyclophosphamide (PCTX), on human and rat granulosa cell function in vitro. In previous experiments we demonstrated that in short-term incubations with rat granulosa cells, high (greater than or equal to 100 micrograms/mL) but not low (less than or equal to 10 micrograms/mL) PCTX concentrations inhibited progesterone accumulation in vitro. In this study, human granulosa cells were obtained from patients undergoing follicle puncture for in vitro fertilization. PCTX at 10, 100, and 500 micrograms/mL, but not at 1 micrograms/mL, resulted in a dose-related reduction in progesterone accumulation in short-term human granulosa cell cultures. Rat granulosa cells were obtained from PMSG-primed immature rats and incubated for 24 h with PCTX concentrations of 1, 5, and 10 micrograms/mL. Ovine LH (10 ng/mL) was added in selected tubes. In comparison to control, progesterone accumulation was significantly reduced by PCTX concentration of 10 micrograms/mL. The above findings demonstrate that cyclophosphamide metabolites, at concentrations achievable in vivo during CTX therapy, decrease rat and human granulosa cell function in vitro. The effect of PCTX on granulosa cells is dependent on PCTX concentration and duration of exposure, as well as the species from which granulosa cells are obtained.