Numerous structurally diverse ligands were developed to target the human histamine H 3 receptor (hH 3 R), a presynaptic G i /G ocoupled auto-and heteroreceptor. Proxyfan was identified to be functionally selective, with different efficacies toward G i /G odependent hH 3 R signaling pathways. However, the underlying molecular mechanism of functional selectivity of proxyfan is still unclear. In the current study, we investigated the role of different G␣ i/o proteins in hH 3 R signaling, using a baculovirus/Sf9 cell expression system. We tested the hypothesis that ligandspecific coupling differences to defined G i /G o -heterotrimers are responsible for functional selectivity of proxyfan at hH 3 R. In Sf9 membranes, full-length hH 3 R (445 amino acids) was expressed in combination with an excess of different mammalian G proteins (G␣ i1 , G␣ i2 , G␣ i3 , or G␣ o1 and ␤ 1 ␥ 2 dimers, respectively). In addition, we constructed the fusion proteins hH 3 R-G␣ i2 and hH 3 R-G␣ o1 to ensure clearly defined receptor/G protein stoichiometries. Steady-state GTPase experiments were performed to directly measure the impact of each G protein on hH 3 R signal transduction. The hH 3 R coupled similarly to all G proteins. We also observed similar ligand-independent or constitutive activity. Proxyfan and various other imidazole-containing ligands, including full agonists, partial agonists, and inverse agonists, showed very similar pharmacological profiles not influenced by the type of G protein coexpressed. Selected ligands, examined in membranes expressing the fusion proteins hH 3 R-G␣ i2 and hH 3 R-G␣ o1 (plus ␤ 1 ␥ 2 dimers), yielded very similar results. Collectively, our data indicate that hH 3 R couples similarly to different G␣ i/o -subunits and that ligand-specific active receptor conformations, resulting in G protein-coupling preferences, do not exist for proxyfan or other imidazole compounds investigated.