Numerous structurally diverse ligands were developed to target the human histamine H(3) receptor (hH(3)R), a presynaptic G(i)/G(o)-coupled auto- and heteroreceptor. Proxyfan was identified to be functionally selective, with different efficacies toward G(i)/G(o)-dependent 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 Galpha(i/o) proteins in hH(3)R signaling, using a baculovirus/Sf9 cell expression system. We tested the hypothesis that ligand-specific 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 (Galpha(i1), Galpha(i2), Galpha(i3), or Galpha(o1) and beta(1)gamma(2) dimers, respectively). In addition, we constructed the fusion proteins hH(3)R-Galpha(i2) and hH(3)R-Galpha(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-Galpha(i2) and hH(3)R-Galpha(o1) (plus beta(1)gamma(2) dimers), yielded very similar results. Collectively, our data indicate that hH(3)R couples similarly to different Galpha(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.