The cloning and analysis of the first identified lysophosphatidic acid (LPA) receptor gene, lpA1 (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 lpB1, lpB2, and lpB3) 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 lpB genes could be grouped into a separate G-protein coupled receptor subfamily, distinct from that containing the LPA receptor genes lpA1 and lpA2. Unlike lpA1 and lpA2, which contain multiple coding exons, all lpB members contained a single coding exon. Heterologous expression of individual lpB members in a hepatoma cell line (RH7777), followed by 35S-GTPgammaS incorporation assays demonstrated that each of the three LPB 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.