To evaluate the effect of reactive oxygen species in human corpus luteum function, we investigated whether hydrogen peroxide (H2O2) affects the in vitro luteal cell production of steroids. H2O2 treatment (1.0-100 microM) of mid and late luteal cell cultures elicited a dose-dependent decrease in basal progesterone production. However, treatment of mid luteal cells with a low concentration of H2O2 (0.01 microM) significantly stimulated progesterone secretion (P < 0.05). In addition, H2O2 (100 microM) markedly inhibited human chorionic gonadotropin (hCG)-stimulated progesterone and estradiol secretion. cAMP production was enhanced (2.4-fold, P < 0.05) by hCG treatment of luteal cells. The addition of H2O2 (0.1-100 microM) to hCG-stimulated luteal cell cultures elicited a decrease in cAMP concentration (P < 0.05) and in the specific binding of radiolabeled hCG by luteal cells. Progesterone and estradiol production stimulated by dibutyryl cAMP were significantly inhibited by H2O2 (P < 0.05). These findings suggest that H2O2 interferes with basal steroid production and, in hCG-stimulated conditions, it may inactivate the gonadotropin-receptor complex. The anti-steroidogenic action of H2O2 therefore raises the possibility of a modulatory role of H2O2 in human luteal steroidogenesis.