GABA type A (GABA(A)) receptors are functionally regulated by external protons in a manner dependent on the receptor subunit composition. Although H(+) can regulate the open probability of single GABA ion channels, exactly what residues and receptor subunits are responsible for proton-induced modulation remain unknown. This study resolves this issue by using recombinant alpha1betai subunit GABA(A) receptors expressed in human embryonic kidney cells. The potentiating effect of low external pH on GABA responses exhibited p(Ka) in accord with the involvement of histidine and/or cysteine residues. The exposure of GABA(A) receptors to the histidine-modifying reagent DEPC ablated regulation by H(+), implicating the involvement of histidine residues rather than cysteines in proton regulation. Site-specific substitution of all conserved external histidines to alanine on the beta subunits revealed that H267 alone, in the TM2 domain, is important for H(+) regulation. These results are interpreted as a direct protonation of H267 on alpha1betai receptors rather than an involvement in signal transduction. The opposing functional effects induced by Zn(2+) and H(+) at this single histidine residue most likely reflect differences in charge delocalization on the imidazole rings in the mouth of the GABA(A) receptor ion channel. Additional substitutions of H267 in beta subunits with other residues possessing charged side chains (glutamate and lysine) reveal that this area of the ion channel can profoundly influence the functional properties of GABA(A) receptors.