Sphingosine 1-phosphate (S1P) stimulates expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells. S1P-induced actions were associated with nuclear factor kappa-B activation and inhibited by pertussis toxin as well as by antisense oligonucleotides specific to S1P receptors, especially, S1P(3). S1P also stimulated endothelial nitric oxide synthase (eNOS) and its activation was markedly inhibited by the antisense oligonucleotide for the S1P(1) receptor rather than that for the S1P(3) receptor. The dose-response curve of S1P to stimulate adhesion molecule expression was shifted to the left in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin and the NOS inhibitor Nomega-nitro-l-arginine methyl ester. NO donor S-nitroso-N-acetylpenicillamine inhibited S1P-induced adhesion molecule expression. Moreover, tumor necrosis factor-alpha-induced adhesion molecule expression was markedly inhibited by S1P in a manner sensitive to inhibitors for PI3-K and NOS. These results suggest that S1P receptors are coupled to both stimulatory and inhibitory pathways for adhesion molecule expression. The stimulatory pathway involves nuclear factor kappa-B and inhibitory one does phosphatidylinositol 3-kinase and NOS.