Sphingosine 1-phosphate (S1P) exerts diverse physiological actions by activating its cognate G protein-coupled receptors. Five S1P receptors have been identified in mammals: LP(B1)/EDG-1, LP(B2)/H218/AGR16/EDG-5, LP(B3)/EDG-3, LP(B4)/NRG-1/EDG-8, and LP(C1)/EDG-6. One of these receptors, LP(B1), has recently been shown to be essential for mouse embryonic development. Here we disrupted the lp(B3) gene in mice, resulting in the complete absence of lp(B3) gene, transcript, and LP(B3) protein. LP(B3)-null mice were viable and fertile and developed normally with no obvious phenotypic abnormality. We prepared mouse embryonic fibroblast (MEF) cells to examine effects of LP(B3) deletion on S1P-induced signal transduction pathways. Wild-type MEF cells expressed lp(B1), lp(B2), and lp(B3) but neither lp(B4) nor lp(C1), and they were highly responsive to S1P in phospholipase C (PLC) activation, adenylyl cyclase inhibition, and Rho activation. Identically prepared LP(B3)-null MEF cells showed significant decreases in PLC activation, slight decreases in adenylyl cyclase inhibition, and no change in Rho activation. Retrovirus-mediated rescue of the LP(B3) receptor in LP(B3)-null MEF cells restored S1P-dependent PLC activation and adenylyl cyclase inhibition. These results indicate a nonessential role for LP(B3) in normal development of mouse but show nonredundant cellular signaling mediated by a single type of S1P receptor.