Sphingosine-1-phosphate (S1P) induces an initial Ca2+-dependent contraction followed by a sustained Ca2+-independent, RhoA-mediated contraction in rabbit gastric smooth muscle cells. The cells coexpress S1P1 and S1P2 receptors, but the signaling pathways initiated by each receptor type and the involvement of one or both receptors in contraction are not known. Lentiviral vectors encoding small interfering RNAs were transiently transfected into cultured smooth muscle cells to silence S1P1 or S1P2 receptors. Phospholipase C (PLC)-β activity and Rho kinase activity were used as markers of pathways mediating initial and sustained contraction, respectively. Silencing of S1P1 receptors abolished S1P-stimulated activation of Gαi3 and partially inhibited activation of Gαi1, whereas silencing of S1P2 receptors abolished activation of Gαq, Gα13, and Gαi2 and partially inhibited activation of Gαi1. Silencing of S1P2 but not S1P1 receptors suppressed S1P-stimulated PLC-β and Rho kinase activities, implying that both signaling pathways were mediated by S1P2 receptors. The results obtained by receptor silencing were corroborated by receptor inactivation. The selective S1P1 receptor agonist SEW2871 did not stimulate PLC-β or Rho kinase activity or induce initial and sustained contraction; when this agonist was used to protect S1P1 receptors so as to enable chemical inactivation of S1P2 receptors, S1P did not elicit contraction, confirming that initial and sustained contraction was mediated by S1P2 receptors. Thus S1P1 and S1P2 receptors are coupled to distinct complements of G proteins. Only S1P2 receptors activate PLC-β and Rho kinase and mediate initial and sustained contraction.