Ovarian follicle atresia is thought to be induced through apoptosis of granulosa cells. This study was designed to investigate the possible involvement of nitric oxide (NO) in granulosa cell apoptosis. In immature rat ovaries obtained 48 h after pregnant mare serum gonadotropin administration, immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), a method to detect apoptotic cells, revealed that inducible NO synthase (iNOS) was predominantly localized in granulosa cells in most healthy immature follicles with TUNEL-negative granulosa cells. In contrast, all atretic follicles with TUNEL-positive granulosa cells were iNOS-negative whatever the developmental stage of the follicle. In cultured granulosa cells, the addition of S-nitroso-N-acetyl-DL-penicillamine (SNAP), an NO generator, directly inhibited spontaneously occurring apoptosis. These results suggest that NO produced by iNOS in granulosa cells of immature follicles may prevent ovarian follicle atresia by inhibiting granulosa cell apoptosis in an autocrine/paracrine manner.