We have previously shown that sphingosine 1-phosphate (S1P) stimulates motility of human umbilical vein endothelial cells (HUVECs) (O.-H. Lee et al., Biochem. Biophys. Res. Commun. 264, 743–750, 1999). To investigate the molecular mechanisms by which S1P stimulates HUVEC motility, we examined tyrosine phosphorylation of p125 focal adhesion kinase (p125^FAK) which is important for cell migration. S1P induces a rapid increase in tyrosine phosphorylation of p125^FAK. Compared with other structurally related lipid metabolites such as sphingosine, C2-ceramide, and lysophosphatidic acid, S1P uniquely stimulated p125^FAK tyrosine phosphorylation and migration of HUVECs. The effect of S1P on p125^FAK tyrosine phosphorylation was markedly reduced by treatment with pertussis toxin or U73122, a phospholipase C (PLC) inhibitor. As a downstream signal of PLC, p125^FAK tyrosine phosphorylation in response to S1P was totally blocked by depletion of the intracellular calcium pool. However, protein kinase C (PKC) inhibitor had no effect on the response to S1P. Finally, chemotaxis assays revealed that inhibition of PLC but not PKC significantly abrogated S1P-stimulated HUVEC migration. These results suggest that the G_i-coupled receptor-mediated PLC-Ca²⁺ signaling pathway may be importantly involved in S1P-stimulated focal adhesion formation and migration of endothelial cells.