|To study the regulation of porcine follicular cell apostosis by gonadotropin, steroid, and nitric oxide, we analyzed DNA fragmentation, the hallmark of apoptosis, and nitrite production of porcine granulosa cells. Dissected individual follicles from ovary were separated in size (small, 2-3 mm; medium 5-6 mm: large, 7-8 mm) and isolated granulosa cells were classified morphologically as atretic or nonatretic. Nitrite concentration was measured by mixing follicular fluids with an equal volume of Griess reagent. Follicular nitric oxide (NO) concentration of healthy follicles was higher than that of atretic follicles. Apoptotic DNA fragmentation was suppressed in non-apoptotic granulosa cells. Follicu-lar apoptosis was induced by androgen but prevented by gonadotropin in vitro. Apoptosis was confined to the granulosa cells. But it was not clear whether apoptosis of granulosa cell was regulated by NO and gonadotropin in vitro. And so non-apoptotic granulosa cells were isolated, incubated with or without gonadotropin, androgen and sodium nitroprusside (SNP), respectively at 37뭖 for 24 hrs. Cultured granulosa cells were used to extract genomic DNA and culture media was assayed for nitrite concen-tration. Nitrite production of culture media was increased, while apoptotic DNA fragmentation was suppressed in PMSG, hCG, testosterone+SNP and SNP treated groups. Nitrite concentration in cul-ture media was decreased, but apoptotic DNA fragmentation was induced in testosterone treated group. These data suggest that NO production and apoptosis may be involved of granulosa cell differentiation and ovarian follicular atresia by gonadotropin. Moreover, NO can overcome the granulosa cell apoptosis induced by testosterone.