Regulation of GABA(A) receptors by extracellular pH exhibits a dependence on the receptor subunit composition. To date, the molecular mechanism responsible for the modulation of GABA(A) receptors at alkaline pH has remained elusive. We report here that the GABA-activated current can be potentiated at pH 8.4 for both alphabeta and alphabeta gamma subunit-containing receptors, but only at GABA concentrations below the EC40. Site-specific mutagenesis revealed that a single lysine residue, K279 in the beta subunit TM2-TM3 linker, was critically important for alkaline pH to modulate the function of both alpha1beta2 and alpha1beta2 gamma2 receptors. The ability of low concentrations of GABA to reveal different pH titration profiles for GABA(A) receptors was also examined at acidic pH. At pH 6.4, GABA activation of alphabeta gamma receptors was enhanced at low GABA concentrations. This effect was ablated by the mutation H267A in the beta subunit. Decreasing the pH further to 5.4 inhibited GABA responses via alphabeta gamma receptors, whereas those responses recorded from alphabeta receptors were potentiated. Inserting homologous beta subunit residues into the gamma2 subunit to recreate, in alphabeta gamma receptors, the proton modulatory profile of alphabeta receptors, established that in the presence of beta2(H267), the mutation gamma2(T294K) was necessary to potentiate the GABA response at pH 5.4. This residue, T294, is homologous to K279 in the beta subunit and suggests that a lysine at this position is an important residue for mediating the allosteric effects of both acidic and alkaline pH changes, rather than forming a direct site for protonation within the GABA(A) receptor.