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.