An orphan G-protein-coupled receptor, GPR18, was cloned on the basis of degenerate-oligonucleotide PCR analysis of HUT 102 cells using primers designed from the conservative regions of the human chemokine receptor. GPR18 was expressed significantly in lymphoid cell lines, but not in non-lymphoid hematopoietic cell lines. Moreover, the expression of the GPR18 gene was higher in peripheral lymphocyte subsets (CD4(+), CD4(+)CD45RA(+), CD4(+)CD45RO(+), CD8(+), and CD19(+)) than in monocytes and lymphoid cell lines, and was increased after stimulation with phytohemagglutinin. By screening using a lipid library, N-arachidonylglycine (NAGly) induced an increase in intracellular Ca(2+) concentration in GPR18-transfected cells, which was significantly greater than that in mock-transfected cells. NAGly also inhibited forskolin-induced cAMP production in a pertussis toxin-sensitive manner in the GPR18-transfected CHO cells. This is the first study to demonstrate that NAGly is a natural ligand for GPR18.