Nitric oxide reacts with superoxide to form peroxynitrite, a potential mediator of oxidant-induced cellular injury. The endothelium is a primary target of injury in many pathological states, including acute lung injury, sepsis, multiple organ failure syndrome, and atherosclerosis, where enhanced production of nitric oxide and superoxide occurs simultaneously. It was hypothesized that stimulation of endothelial cell nitric oxide production would result in formation of peroxynitrite. Immediate oxidant production was detected by luminol- and lucigenin-enhanced chemiluminescence from cultured bovine aortic endothelial cells exposed to bradykinin or to the calcium ionophore A23187. Luminol-enhanced chemiluminescence was efficiently inhibited by the nitric oxide synthase inhibitor nitro-L-arginine methyl ester and by superoxide dismutase, implying dependence on the presence of both nitric oxide and superoxide for oxidant production. Inhibition of luminol-enhanced chemiluminescence by nitro-L-arginine methyl ester was partially reversed by L-arginine, but not by D-arginine. Cysteine, methionine, and urate, known inhibitors of peroxynitrite-mediated oxidation, inhibited luminol-enhanced chemiluminescence, while the hydroxyl radical scavengers, mannitol and dimethylsulfoxide, and catalase did not. Bicarbonate increased luminol-enhanced chemiluminescence in a concentration-dependent manner. Superoxide production, detected by lucigenin-enhanced chemiluminescence, was slightly increased in the presence of nitro-l-arginine methyl ester, suggesting that endothelial cell-produced superoxide was partially metabolized by reaction with nitric oxide. These results are consistent with agonist-induced peroxynitrite production by endothelial cells and suggests that peroxynitrite may have an important role in oxidant-induced endothelial injury.