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 μm) 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 μm) significantly stimulated progesterone secretion (P<0·05). In addition, H2O2 (100 μm) 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 μm) 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.