In a steady-state GTPase activity assay, N-[3-(1H-imidazol-4-yl) propyl)]guanidines and N-G-acylated derivatives are more potent and efficacious at fusion proteins of guinea pig (gpH(2)R-G(s alpha S)) than human (hH(2)R-G(s alpha S)) histamine H-2 receptor, coupled to the short splice variant of G(s alpha), G(s alpha S). Whereas Ala-271 (hH(2)R) and Asp-271 (gpH(2)R) in transmembrane domain 7 were identified to determine the potency differences of guanidine-type agonists, the molecular basis for the efficacy differences remains to be elucidated. A homology model of the gpH2R suggested that an H-bond between Tyr-17 and Asp-271 stabilizes an active receptor conformation of the gpH2R. In the present study, we generated a mutant hH(2)R-G(s alpha S) with Cys-17-->Tyr-17/Ala-271-->Asp-271 exchanges (hH(2)R-->gpH(2)R) that exhibited an enhanced level of constitutive GTPase activity and adenylyl cyclase activity compared with wild-type hH(2)R-G(s alpha S) and gpH(2)R-G(s alpha S). Potencies and efficacies of guanidines and N-G-acylguanidines were increased at this mutant receptor compared with hH(2)R-Gs alpha S, but they were still lower than at gpH(2)R-G(s alpha S), suggesting that aside from Tyr-17 and Asp-271 additional amino acids contribute to the distinct pharmacological profiles of both species isoforms. Another hH(2)R-G(s alpha S) mutant with a Cys-17-->Tyr-17 exchange showed inefficient coupling to G(s alpha S) as revealed by reduced agonist-stimulated GTPase and basal adenylyl cyclase activities. Collectively, our present pharmacological study confirms the existence of an H-bond between Tyr-17 and Asp-271 favoring the stabilization of an active receptor conformation. Distinct potencies and efficacies of agonists and inverse agonists further support the concept of ligand-specific conformations in wild-type and mutant H2R-G(s alpha S) fusion proteins.