We previously showed in in vivo and in vitro studies that nitric oxide (NO) is engaged in luteolysis in cattle. Nitric oxide produced locally in the bovine corpus luteum (CL) inhibits progesterone (P4) synthesis and is suggested to be a component of the luteolytic cascade induced by uterine prostaglandin (PG) F2alpha. In the present study, the molecular mechanisms of NO action during structural luteolysis were studied in cultured bovine luteal cells (Days 15-17 of the estrous cycle). The effects of the NO donor (NONOate; 10(-4)M) on DNA fragmentation, cell viability, P4 production and caspase-3 activity were compared with those of PGF2alpha (10(-6)M). Moreover, mobilization of intracellular calcium [Ca2+]i and gene expressions of Fas-L, Fas, bcl-2, bax, and caspase-3 in the cells were determined by semi-quantitative RT-PCR after NONOate treatment. Caspase-3 activity was examined calorimetrically. Contrary to PGF2alpha NONOate decreased cell viability. DNA fragmentation after NONOate treatment increased by more than with PGF22alpha. NONOate increased mobilization of [Ca2+]i in the cells. Although the NO donor did not affect Fas-L and bcl-2 gene expression, it stimulated Fas and bax mRNA and caspase-3 expression. The ratio of bcl-2 to bax mRNA level decreased in the cells treated with NONOate. Moreover, NONOate stimulated caspase-3 activity more effectively than PGF2alpha. The overall results suggest that NO is a luteolytic factor that plays a crucial role in regulation of the estrous cycle in structural luteolysis by inducing apoptosis of luteal cells in cattle.