We have previously shown that extracellular protons inhibit recombinant and native GABA_A receptors. In the present report, we studied the site(s) and mechanism by which protons modulate the GABA_A receptor. Whole-cell GABA-activated currents were recorded from human embryonic kidney (HEK) 263 cells expressing recombinant 122 GABA_A 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 1(L264T)22 receptors, suggesting pH does not modulate channel activity by affecting the channel gating process directly. To test the hypothesis that protons modulate GABA_A 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 2 Y205 to F or 1 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 H⁺ and Zn²⁺ do not share a common site of action. Additional experiments further tested this possibility. Treatment with the histidine-modifying reagent diethlpyrocarbonate (DEPC) reduced Zn²⁺-mediated inhibition of GABA_A receptors, but had no effect on proton-induced inhibition of GABA currents. In addition, mutations of residues known to be involved in Zn²⁺ modulation had no effect on pH modulation of GABA_A receptors. Our results support the hypothesis that protons inhibit GABA_A receptor function by direct or allosteric interaction with the GABA binding site. In addition, the sites of action of H⁺ and Zn²⁺ in GABA_A receptors are distinct.