Ceramide is a novel second messenger generated by hydrolysis of membrane sphingomyelin by a neutral sphingomyelinase (nSMase). Cytokines such as tumor necrosis factor- (TNF-) have been shown to increase intracellular ceramide through phospholipase A₂ (PLA₂)-dependent activation of nSMase. TNF- has been shown to cause endothelium-independent relaxation in isolated blood vessels. We have previously shown that exogenously applied sphingomyelinase and ceramide cause endothelium-independent vasodilation in rat thoracic aortas (D. G. Johns, H. Osborn, and R. C. Webb. Biochem. Biophys. Res. Commun. 237: 95-97, 1997). In the present study, we tested the hypothesis that ceramide mediates TNF--induced vasodilation. In phenylephrine-contracted rat thoracic aortic rings (no endothelium), TNF- caused concentration-dependent relaxation in the presence of cyclooxygenase and lipoxygenase inhibitors. The phospholipase A₂ antagonist 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid (DEDA; 50 µM) and the nonselective PLA₂ antagonist quinacrine (30 µM) inhibited TNF--induced relaxation. In cultured rat aortic vascular smooth muscle cells, TNF- (10⁷ g/ml) increased intracellular ceramide 1.5-fold over basal level (0.08 nmol/mg protein), which was blocked by the PLA₂ antagonist DEDA (50 µM). We conclude that PLA₂ activation and increased ceramide generation play a role in mediating TNF--induced endothelium-independent vasodilation.