We speculated that the sphingosine-1-phosphate (S1P) receptor S1P₂, which uniquely inhibits cell migration, might mediate inhibitory effects on endothelial cell migration and angiogenesis, different from S1P₁ and S1P₃. Mouse vascular endothelial cells, which endogenously express S1P₂ and S1P₃, but not S1P₁, responded to S1P and epidermal growth factor (EGF) with stimulation of Rac, migration, and the formation of tube-like structures on the Matrigel. The S1P₃-antagonist VPC-23019 abolished S1P-induced, G_i-dependent Rac stimulation, cell migration, and tube formation, whereas the S1P₂-antagonist JTE-013 enhanced these S1P-induced responses, suggesting that S1P₂ exerts inhibitory effects on endothelial Rac, migration, and angiogenesis. S1P₂ overexpression markedly augmented S1P-induced, G_i-independent inhibition of EGF-induced migration and tube formation. Finally, the blockade of S1P₂ by JTE-013 potentiated S1P-induced stimulation of angiogenesis in vivo in the Matrigel implant assay. These observations indicate that in contrast to S1P₁ and S1P₃, S1P₂ negatively regulates endothelial morphogenesis and angiogenesis most likely through down-regulating Rac.