Injection of a luteolytic dose of prostaglandin (PG) F2α induces an acute increase in plasma concentration of nitric oxide (NO) metabolites (nitrite/nitrate). Furthermore, NO suppresses progesterone (P4) production and induces apoptosis in bovine luteal cells. These findings suggest that NO plays important roles in luteolysis. However, the regulation of NO generating system in bovine CL is not fully understood. Although tumor necrosis factor α (TNF) and interferon γ (IFNG) produced by immune cells have been demonstrated stimulate NO production in extragonadal cells, the roles of TNF and IFNG in the regulation of NO production, NO synthase (NOS) expression and NOS activity in the bovine corpus luteum (CL) remain unclear. To elucidate whether NO generating system in luteal endothelial cells (LECs) is regulated by cytokines, the effects of TNF or IFNG on the expression of NOS (inducible NO synthase; iNOS and endothelial NO synthase; eNOS) mRNA, protein and NO production were examined. In addition, the direct effects of estrogen (E2) and P4 on NOS proteins and NOS activity were examined. LECs were obtained from mid CL (Days 8-12 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. Cultured bovine LECs were treated for 24 h with TNF (2.9 nM), IFNG (2.5 nM), E2 (10 nM) or P4 (1 μM). Both TNF and IFNG stimulated iNOS mRNA, protein expression and NO production. Furthermore, E2 stimulatediNOS protein expression, whereas P4 inhibited iNOS protein expression. In contrast to iNOS, eNOS protein expression was not affected by treatments with TNF, IFNG, E2 or P4. By observing the conversion of [3C]L-arginine to [3C]L-citrulline, we found that TNF, IFNG and E2 stimulated the NOS activity in cultured LECs (P<0.05). The overall results demonstrate that TNF, IFNG and E2 accelerate NO production by stimulating iNOS protein expression as well as NOS activity. On the other hand, P4 was able to suppress iNOS expression in LECs. This research was supported by JSPS Grants (No. 19580326 and No. 18380166), and by a research fund from the Livestock Technology Association (LTA), Japan. (poster)