Histamine is an important neurotransmitter that exerts its physiological actions through H1-4 metabotropic receptors in mammals. It also directly activates ionotropic GABAA receptor (GABAAR) β3 homooligomers and potentiates GABA responses in αβ heterooligomers in vitro, but the respective histamine binding sites in GABAARs are unknown. We hypothesized that histamine binds at the extracellular β+β– interface at a position homologous to the GABA binding site of heterooligomeric GABAARs. To test this, we individually mutated several residues at the putative ligand binding minus side of a rat GABAAR β3 wild type subunit and of a β3 subunit that was made insensitive to trace Zn2+ inhibition [β3(H267A); called Zβ3]. Zβ3, Zβ3(Y62L), Zβ3(Q64A), Zβ3(Q64E), α1Zβ3, or α1Zβ3(Y62L) receptors were studied in HEK293T cells using whole cell voltage clamp recording. β3, β3(Y62C), β3(Q64C), β3(N41C), β3(D43C), β3(A45C) or β3(M115C) receptors were examined in Xenopus oocytes using two-electrode voltage clamp. Histamine directly activated Zβ3 and β3 homooligomers and potentiated GABA actions in α1Zβ3 heterooligomers. Receptors containing Zβ3(Y62L), β3(Y62C) and β3(D43C) showed markedly reduced histamine potency, but homo- and heterooligomers with Zβ3(Q64E) exhibited increased potency. The GABAAR αβ(γ) competitive antagonist bicuculline elicited sub-maximal agonist currents through Zβ3 homooligomers, the potency of which was strongly decreased by Zβ3(Y62L). Mutations β3(N41C), β3(A45C) and β3(M115C) disturbed receptor expression or assembly. Computational docking into the crystal structure of homooligomeric β3 receptors resulted in a histamine pose highly consistent with the experimental findings, suggesting that histamine activates β3 receptors via a site homologous to the GABA site in αβγ receptors.