Recent evidence suggested that nitric oxide (NO) acts as an important factor in a variety of physiological and pathological roles, including reproductive functions. The purpose of the present study was to investigate whether NO might significantly induce any change in steroidogenesis in cultured porcine granulosa cells (PGC). An NO donor, NOC18, significantly suppressed the oestradiol release from basal (unstimulated) and gonadotrophin-stimulated PGC in a 2 h culture. NOC18 also significantly inhibited the aromatase activity of basal and gonadotrophin-stimulated PGC as measured by a modified tritiated water method. However, the cGMP analogue, 8-bromo-cGMP, had no significant effect on the accumulation of oestradiol and progesterone in basal and gonadotrophin-stimulated PGC during 24 h culture. An NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine (LNMMA), significantly stimulated the basal oestradiol release and dose-dependently enhanced the oestradiol and progesterone release from follicle stimulating hormone (FSH)-stimulated PGC in a 24 h culture. However, NG-monomethyl-D-arginine, which does not inhibit NOS, did not enhance the release of oestradiol and progesterone under the same experimental conditions. LNMMA also significantly suppressed the nitrite concentrations in the media as measured by chemiluminescence. These results demonstrate for the first time that NO inhibits oestradiol secretion independent of cGMP by inhibiting P450 aromatase activity in moderately mature PGC.