Previous studies revealed pharmacological differences between human and guinea pig histamine H-2 receptors (H(2)Rs) with respect to the interaction with guanidine-type agonists. Because H2R species variants are structurally very similar, comparative studies are suited to relate different properties of H2R species isoforms to few molecular determinants. Therefore, we systematically compared H(2)Rs of human (h), guinea pig (gp), rat (r), and canine (c). Fusion proteins of hH(2)R, gpH(2)R, rH(2)R, and cH(2)R, respectively, and the short splice variant of G(s alpha), G(s alpha S), were expressed in Sf9 insect cells. In the membrane steady-state GTPase activity assay, cH(2)R-G(s alpha S) but neither gpH(2)R-G(s alpha S) nor rH(2)R-G(s alpha S) showed the hallmarks of increased constitutive activity compared with hH(2)R-G(s alpha S), i.e., increased efficacies of partial agonists, increased potencies of agonists with the extent of potency increase being correlated with the corresponding efficacies at hH(2)R-G(s alpha S), increased inverse agonist efficacies, and decreased potencies of antagonists. Furthermore, in membranes expressing nonfused H(2)Rs without or together with mammalian G(s alpha S) or H2R-G(s alpha) fusion proteins, the highest basal and GTP-dependent increases in adenylyl cyclase activity were observed for cH(2)R. An example of ligand selectivity is given by metiamide, acting as an inverse agonist at hH(2)R-G(s alpha S), gpH(2)R-G(s alpha S), and rH(2)R-G(s alpha S) in the GTPase assay in contrast to being a weak partial agonist with decreased potency at cH(2)R-G(s alpha S). In conclusion, the cH(2)R exhibits increased constitutive activity compared with hH(2)R, gpH(2)R, and rH(2)R, and there is evidence for ligand-specific conformations in H2R species isoforms.