1. A few studies have shown that nitric oxide may exert cytotoxic and/or steroidogenic effects on cultured ovarian cells but the source of this factor within the ovary remains equivocal. 2. In this study we have investigated the effects of nitric oxide on progesterone secretion, cell viability and cell morphology of cultured rat granulosa/lutein cells and examined whether granulosa cells are an important source of nitric oxide. 3. Only very low or undetectable levels of nitrites were measured in granulosa/lutein-cell-only cultures, although there was a small but significant increase in nitrite release observed in granulosa/ lutein cells obtained from oestrous rats compared with those obtained from proestrous rats. 4. There was a concentration-dependent inhibition of progesterone synthesis in the presence of the nitric oxide donors sodium nitroprusside and S-nitroso-N-acetyl-penicillamine which corresponded with an increased concentration of nitrite accumulation in the culture medium. 5. High concentrations of nitrites were measured in the medium of granulosa/lutein cells co-cultured with peritoneal macrophages and progesterone synthesis was inhibited. This effect of the macrophages was partially reversed by inhibitors of nitric oxide synthesis, aminoguanidine, NG-methyl-L-arginine and NG-L-arginine-methyl-ester, and the reversal of inhibition was inversely proportional to the concentration of nitrites measured in the medium. Dose-response curves for the three drugs on the inhibition of nitrite accumulation in macrophage cultures were obtained. 6. The nitric oxide scavenger c-PTIO [2-(4-carboxy-phenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide.potassium salt] partially reversed the effects of S-nitroso-N-acetyl-penicillamine and macrophages on progesterone synthesis in granulosa/ lutein cells. 7. With the exception of the high dose of sodium nitroprusside, there was no evidence that any of the drugs reduced cellular viability, as assessed by measurement of cellular dehydrogenases and Trypan Blue exclusion, although high concentrations of nitrite in the culture medium derived either from the nitric oxide donors or macrophages were associated with a loss of morphological luteinization. 8. Based on the available evidence, we suggest that nitric oxide can inhibit steroidogenesis and that in vivo the source of nitric oxide may be from macrophages, which invade the ovary during the periovulatory period, and/or from endothelial cells of ovarian blood vessels.