We speculated that the sphingosine-1-phosphate (S1P) receptor S1P 2 , which uniquely inhibits cell migration, might mediate inhibitory effects on endothelial cell migration and angiogenesis, different from S1P 1 and S1P 3 . Mouse vascular endothelial cells, which endogenously express S1P 2 and S1P 3 , but not S1P 1 , responded to S1P and epidermal growth factor (EGF) with stimulation of Rac, migration, and the formation of tube-like structures on the Matrigel. The S1P 3 -antagonist VPC-23019 abolished S1P-induced, G i -dependent Rac stimulation, cell migration, and tube formation, whereas the S1P 2 -antagonist JTE-013 enhanced these S1P-induced responses, suggesting that S1P 2 exerts inhibitory effects on endothelial Rac, migration, and angiogenesis. S1P 2 overexpression markedly augmented S1P-induced, G i -independent inhibition of EGF-induced migration and tube formation. Finally, the blockade of S1P 2 by JTE-013 potentiated S1P-induced stimulation of angiogenesis in vivo in the Matrigel implant assay. These observations indicate that in contrast to S1P 1 and S1P 3 , S1P 2 negatively regulates endothelial morphogenesis and angiogenesis most likely through down-regulating Rac.