A hallmark of luteolysis is leukocyte infiltration. Phagocytic leukocytes are well known to evoke a burst release of hydrogen peroxide (H2O2) of sufficient magnitude to injure cells. We, therefore, evaluated the effect of H2O2 in isolated rat luteal cells. Peroxide (100 microM; the near-half-maximal dose) markedly inhibited both LH-sensitive cAMP accumulation and progesterone production within 5 min of treatment. Cell levels of ATP were also reduced by H2O2, but not until 10 min after exposure, with more than 50% depletion within 60 min. This depletion of ATP by H2O2 was prevented by nicotinamide or 3-aminobenzamide, inhibitors of DNA repair, but these inhibitors did not prevent the antigonadotropic action of H2O2. cAMP accumulation in response to forskolin was not inhibited by H2O2 when ATP depletion was blocked with 3-aminobenzamide. The specific binding of radiolabeled hCG to isolated cells or the biological activity of LH was not affected by H2O2 pretreatment of the cells. Isobutylmethylxanthine had no effect on abrogation of LH-sensitive cAMP accumulation by H2O2. The actions of H2O2 were not mediated by prostaglandin, since indomethacin was without effect and an increase in prostaglandin F2 alpha production was not seen with H2O2 treatment. The acute luteolytic actions of H2O2 thus appear to be due to a very rapid desensitization of the LH-receptor complex, followed by depletion of ATP. The abrogation of luteotropic support and the injurious consequences of ATP depletion raise the possibility that H2O2 may be a physiological mediator of luteolysis.