The present study tested the hypothesis that ceramide, a sphingomylinase metabolite, serves as an second messenger for TNF- to stimulate superoxide production, thereby decreasing endothelium-dependent vasorelaxation in coronary arteries. In isolated bovine small coronary arteries, TNF- (1 ng/ml) markedly attenuated vasodilator responses to bradykinin and A23187. In the presence of N^G-nitro-L-arginine methyl ester, TNF- produced no further inhibition on the vasorelaxation induced by these vasodilators. Using DAF-2 DA fluorescence imaging analysis, bradykinin was found to increase nitric oxide (NO) concentrations in the endothelium of isolated bovine small coronary arteries, which was inhibited by TNF-. Pretreatment of the arteries with desipramine (10 µM), an inhibitor of acidic sphingomyelinase, tiron (1 mM), a superoxide scavenger and polyethylene glycol superoxide dismutase (100 U/ml), largely restored the inhibitory effect of TNF- on bradykinin- and A23187-induced vasorelaxation. In addition, TNF- activated acidic sphingomyelinase and increased ceramide levels in coronary endothelial cells. We conclude that TNF- inhibits NO-mediated endothelium-dependent vasorelaxation in small coronary arteries via sphingomyelinase activation and consequent superoxide production in endothelial cells.