The role of eicosanoids in atherogenesis has not been thouroughly explained. This is partly due to the numerous eicosanoids and the variable effects that each has on different systems. Apoptosis of vascular smooth muscle cells has been shown to play a role in the atherosclerotic disease leading to lesion formation and further destabilization of the formed lesion. In this study, we have investigated the role of arachidonic acid derived eicosanoids in nitric oxide (NO)-stimulated vascular smooth muscle cells. We have shown previously that the nitric oxide (NO)-induced apoptosis of vascular smooth muscle cells was accompanied by arachidonic acid release via cytoplasmic phospholipase A2 (cPLA2) activation. Also, arachidonic acid, but not oleic acid, induced apoptosis of these cells at low concentrations (5–10 μM). Our results revealed that the cPLA2 specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), blocked NO-induced eicosanoid production, while the presence of arachidonic acid enhanced the ability of the cells to make prostaglandin E2 (PGE2). Also, inhibitors of the cyclo-oxygenase (Cox) enzymes, such as N-[2-cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398), a specific Cox-2 inhibitor, or indomethacin, a non-specific Cox inhibitor, blocked NO-induced PGE2 production and apoptosis of vascular smooth muscle cells to the same extent, indicating that apoptosis might be induced by a Cox-2 metabolic product. In addition to these observations, the eicosanoids investigated, namely, PGE2, PGI2 LTB4, and PGJ2, showed different effects on vascular smooth muscle cells. Both PGJ2 and LTB4 decreased the percentage of viable cells and induced apoptosis of vascular smooth muscle cells, while PGE2 and PGI2 had no effect on cell viability and failed to induce apoptosis. These data suggest that eicosanoids, such as PGJ2, but not PGE2 or PGI2, are involved in NO-induced apoptosis of vascular smooth muscle cells and that the eicosanoid synthesis pathways might be utilized for vascular therapeutic strategies. © 2005 Wiley-Liss, Inc.