The endothelial differentiation gene-6 (Edg-6) was recently identified as an orphan G-protein-coupled receptor. Its predicted amino acid sequence is very close to Edg family of receptor proteins whose ligand is supposed to be lysophosphatidic acid (LPA) or lysosphingolipid such as sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), Transfection of the Edg-6 into Chinese hamster ovary (CHO) cells and K562 cells resulted in the appearance of high-affinity [H-3]SIP binding activity. Among lipids employed, S1P and, even though less potent, SPC, displaced the [H-3]S1P binding, but LPA was inactive. In Edg-6-transfected CHO cells, an increase in cytosolic Ca2+ concentration in response to S1P or SPC was clearly enhanced without change in the LPA-induced action as compared with the vector-transfected cells. The enhancement of the Ca2+ response was associated with a significant accumulation of inositol phosphate, reflecting activation of phospholipase C. Similar enhancement of Ca2+ response to S1P or SPC was also observed in Edg-6-expressing K562 cells. These lipid-induced actions in CHO cells and K562 cells expressing Edg-6 were markedly suppressed by pertussis toxin treatment. We conclude that Edg-6 is one of S1P or lysosphingolipid receptors that couple to phospholipase C-Ca2+ system through pertussis toxin-sensitive G-proteins, 2000 Academic Press.