The aim of the present report was to investigate the ligand selectivity of the human orphan G-protein-coupled receptor GPR100 (hGPR100), recently identified as a novel bradykinin (BK) receptor, as compared with that of the human B(2) receptor (hB(2)R) stably transfected in Chinese hamster ovary cells. BK was able to inhibit the cAMP production induced by forskolin with a potency 100-fold lower at the hGPR100 (pEC(50) = 6.6) than that measured at the hB(2)R (pEC(50) = 8.6). Both effects were inhibited by the B(2) receptor antagonist Icatibant (1 microM). The nonpeptide B(2) receptor agonist FR190997 (8-[2,6-dichloro-3-[N-methylcarbamoyl)cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline) did inhibit the forskolin-induced cAMP production (pEC(50) = 7.7) at the hB(2)R, whereas it was not able to exert any effect at the hGPR100. The human insulin-like peptide relaxin 3 did inhibit the cAMP production at the hGPR100 (pEC(50) = 7.3) at a greater extent than BK, and was devoid of any effect at the hB(2)R. FR190997 and relaxin 3 responses at the hB(2)R and hGPR100, respectively, were not inhibited by Icatibant (1 microM). These data indicate FR190997 and relaxin 3 as selective agonists for hB(2)R and hGPR100, respectively, and support the concept that different agonists may specifically bias the conformational states of a receptor to result in a final common G protein coupling, which is differentially recognized by antagonists.