Ligand pharmacology of histamine H 3 -receptors is species-dependent. In previous studies, two amino acids in transmembrane domain 3 (TM III) were shown to play a significant role. In this study, we characterized human and rat histamine H 3 -receptors (hH 3 R and rH 3 R, respectively), co-expressed with mammalian G proteins in Sf9 insect cell membranes. We compared a series of imidazole-containing H 3 R ligands in radioligand binding and steady-state GTPase assays. H 3 Rs similarly coupled to Gα i/o -proteins. Affinities and potencies of the agonists histamine, N α -methylhistamine and R-(α)-methylhistamine were in the same range. Imetit was only a partial agonist. The pharmacology of imetit and proxifan was similar at both species. However, impentamine was more potent and efficacious at rH 3 R. The inverse agonists ciproxifan and thioperamide showed higher potency but lower efficacy at rH 3 R. Clobenpropit was not species-selective. Strikingly, imoproxifan was almost full agonist at hH 3 R, but an inverse agonist at rH 3 R. Imoproxifan was docked into the binding pocket of inactive and active hH 3 R- and rH 3 R-models and molecular dynamic simulations were performed. Imoproxifan bound to hH 3 R and rH 3 R in E-configuration, which represents the trans-isomer of the oxime-moiety as determined in crystallization studies, and stabilized active hH 3 R-, but inactive rH 3 R-conformations. Large differences in electrostatic surfaces between TM III and TM V cause differential orientation of the oxime-moiety of imoproxifan, which then differently interacts with the rotamer toggle switch Trp 6.48 in TM VI. Collectively, the substantial species differences at H 3 Rs are explained at a molecular level by the use of novel H 3 R active-state models.