The signaling pathways that couple tumor necrosis factor-alpha (TNFalpha) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFalpha induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFalpha resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFalpha on endothelial cells leading to extracellular signal-regulated kinases and NF-kappaB activation, whereas ceramide or sphingosine was not. Furthermore, N, N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFalpha-induced extracellular signal-regulated kinases and NF-kappaB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFalpha-induced endothelial cell activation.