Extracellular pH regulates glycine receptors through an unknown mechanism. Here we demonstrate that acidic pH remarkably inhibited glycine-activated whole-cell currents in recombinant glycine α1 and α1β receptors transiently expressed in human embryonic kidney 293 cells. The proton effect was voltage-independent and pharmacologically competed with glycine receptor agonist glycine and antagonist strychnine. Using site-directed mutagenesis, we have identified an N-terminal domain that is essential for proton-induced inhibition of glycine current. In α1 homomers, removal of the hydroxyl group by mutation of residue Thr-112 to Ala or Phe abolished inhibition of glycine currents by acidification. In contrast, mutation of Thr-112 to another hydroxylated residue (Tyr) produced receptors that retained partial proton sensitivity. In α1β heteromers, a single mutation of the 13 subunit T135A, which is homologous to α1 Thr-112, reduced proton sensitivity, whereas the double mutation α1(T112A)β(T135A) almost completely eliminated the proton sensitivity. In addition, the mutation α1 H109A greatly reduced sensitivity to protons in homomeric α1 receptors. The results demonstrate that extracellular pH can regulate the function of glycine α1 and α1β receptors. An extracellular domain consisting of Thr-112 and His-109 at the α1 subunit and Thr-135 at the β subunit plays a critical role in determining proton modulation of glycine receptor function.