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 co-express 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 (siRNAs) were transiently transfected into cultured smooth muscle cells to silence S1P₁ or S1P₂ receptors. Phospholipase C (PLC)- 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 G_i3 and partially inhibited activation of G_i1, whereas silencing of S1P₂ receptors abolished activation of G_q, G₁₃, and G_i2, and partially inhibited activation of G_i1. Silencing of S1P₂ but not S1P₁ receptors suppressed S1P-stitmulated PLC- 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- 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- and Rho kinase and mediate initial and sustained contraction.