The cloning and analysis of the first identified lysophosphatidic acid (LPA) receptor gene, lp_A1 (also referred to as vzg-1 or edg-2), led us to identify homologous murine genes that might also encode receptors for related lysophospholipid ligands. Three murine genomic clones (designated lp_B1, lp_B2, and lp_B3) were isolated, corresponding to human/rat Edg-1, rat H218/AGR16, and human edg-3, respectively. Based on the amino acid similarities of their predicted proteins (44–52% identical), the three lp_B genes could be grouped into a separate G-protein coupled receptor subfamily, distinct from that containing the LPA receptor genes lp_A1 and lp_A2. Unlike lp_A1 and lp_A2, which contain multiple coding exons, all lp_B members contained a single coding exon. Heterologous expression of individual lp_B members in a hepatoma cell line (RH7777), followed by ³⁵S-GTPγS incorporation assays demonstrated that each of the three LP_B receptors conferred sphingosine-1-phosphate-dependent, but not lysophosphatidic acid-dependent, G-protein activation. Northern blot and in situ hybridization analyses revealed overlapping as well as distinct expression patterns in both embryonic and adult tissues. This comparative characterization of multiple sphingosine-1-phosphate receptor genes and their spatiotemporal expression patterns will aid in understanding the biological roles of this enlarging lysophospholipid receptor family.