We investigated the presence of nitric oxide (NO) synthase in ovarian follicular cells obtained from women undergoing in vitro fertilization procedures. Endothelial NO synthase messenger RNA was demonstrated by polymerase chain reaction amplification of reverse transcribed RNA. NO synthase was localized to granulosa-luteal cells by immunocytochemistry, using a monoclonal antibody. Ovarian follicular cell NO synthase enzyme activity was confirmed by measuring the conversion of L-arginine to citrulline. To investigate the effect of NO on granulosa-luteal cell steroidogenesis, NO synthase inhibitors and NO donors were added to cell cultures. NG-Monomethyl-L-arginine and N-nitro-arginase methyl ester, selective inhibitors of NO synthase, significantly increased estradiol secretion by granulosa-luteal cells. S-Nitroso-L-acetyl penicillamine (S-NAP) and S-nitroso glutathione, NO donors, caused a dose-dependent decrease in both estradiol and progesterone secretion. The decrease by S-NAP was reversed by hemoglobin, which binds free NO. Although S-NAP increased the concentration of cGMP in granulosa-luteal cells, cGMP analogs had no effect on steroidogenesis in cell cultures. S-NAP and native NO in solution decreased cellular and microsomal aromatase activities. We conclude that NO synthase is present in human granulosa-luteal cells and that NO inhibits estradiol secretion independent of cGMP by directly inhibiting aromatase.