We previously identified H109 of the glycine alpha1 subunit as a putative proton binding site. In the present studies, we explored additional proton binding site(s) as well as the mechanism underlying modulation of glycine receptors by protons. Whole-cell glycine currents were recorded from HEK 293 cells transiently expressing wild type or mutant glycine receptors. Individual mutation of 3 of 4 remaining extracellular histidine residue into alanine (i.e., alpha1 H107A, H215A or H419A), reduced the receptor sensitivity to protons to a varying extent. In contrast, mutation of alpha1 H201A did not affect proton sensitivity. Double, triple or quadruple histidine mutation of these residues caused a further reduction of proton sensitivity, suggesting multiple binding sites for proton action on glycine receptors. Furthermore, the substitution T133A, which mediates Zn(2+) inhibition, virtually abolished the proton effect on peak amplitude and current kinetics of glycine response. Replacement of T with S on position 133 partially restored receptor sensitivity to protons, suggesting the hydroxyl group of residue T133 is essential for proton-mediated modulation. In heteromeric alpha1beta receptors, mutations beta H132A and S156A, which correspond to H109 and T133 of the alpha1 subunit, respectively, also affected proton inhibition. In conclusion, multiple extracellular histidine residues (H107, H109, H215 and H419) and threonine residues of the alpha1 and beta Zn(2+) coordination sites are critical for modulation of the glycine receptor by protons.