Five cognate G protein-coupled receptors (S1P(1-5)) have been shown to mediate various cellular effects of sphingosine 1-phosphate (S1P). Here we report the generation of mice null for S1P(2) and for both S1P(2) and S1P(3). S1P(2)-null mice were viable and fertile and developed normally. The litter sizes from S1P(2)S1P(3) double-null crosses were remarkably reduced compared with controls, and double-null pups often did not survive through infancy, although double-null survivors lacked any obvious phenotype. Mouse embryonic fibroblasts (MEFs) were examined for the effects of receptor deletions on S1P signaling pathways. Wild-type MEFs were responsive to S1P in activation of Rho and phospholipase C (PLC), intracellular calcium mobilization, and inhibition of forskolin-activated adenylyl cyclase. S1P(2)-null MEFs showed a significant decrease in Rho activation, but no effect on PLC activation, calcium mobilization, or adenylyl cyclase inhibition. Double-null MEFs displayed a complete loss of Rho activation and a significant decrease in PLC activation and calcium mobilization, with no effect on adenylyl cyclase inhibition. These data extend our previous findings on S1P(3)-null mice and indicate preferential coupling of the S1P(2) and S1P(3) receptors to Rho and PLC/Ca(2+) pathways, respectively. Although either receptor subtype supports embryonic development, deletion of both produces marked perinatal lethality, demonstrating an essential role for combined S1P signaling by these receptors.