We have previously shown that extracellular protons inhibit recombinant and native GABAA receptors. In this report, we studied the site(s) and mechanism by which protons modulate the GABAA receptor. Whole cell GABA-activated currents were recorded from human embryonic kidney (HEK) 293 cells expressing recombinant α1β2γ2 GABAA receptors. Protons competitively inhibited the response to GABA and bicuculline. In contrast, change in pH did not influence direct gating of the channel by pentobarbital, and it did not influence spontaneous channel openings in α1(L264T)β2γ2 receptors, suggesting pH does not modulate channel activity by affecting the channel gating process directly. To test the hypothesis that protons modulate GABAA receptors at the ligand binding site, we systemically mutated N-terminal residues known to be involved in GABA binding and assessed effects of pH on these mutant receptors. Site-specific mutation of β2 Y205 to F or α1 F64 to A, both of which are known to influence GABA binding, significantly reduced pH sensitivity of the GABA response. These mutations did not affect Zn2+ sensitivity, suggesting that H+ and Zn2+ do not share a common site of action. Additional experiments further tested this possibility. Treatment with the histidine-modifying reagent diethylpyrocarbonate (DEPC) reduced Zn 2+-mediated inhibition of GABAA receptors but had no effect on proton-induced inhibition of GABA currents. In addition, mutation of residues known to be involved in Zn2+ modulation had no effect on pH modulation of GABAA receptors. Our results support the hypothesis that protons inhibit GABAA receptor function by direct or allosteric interaction with the GABA binding site. In addition, the sites of action of H+ and Zn2+ in GABAA receptors are distinct.