Ligand-gated ion channels of the cys-loop receptor family have received considerable attention as putative sites of action causing the behavioral effects of ethanol. Studies over the last decade identified several positions in the transmembrane domain critical for ethanol modulation of glycine receptors (GlyRs). Studies to date in the extracellular domain of alpha1GlyRs found that mutations at position 52 in Loop 2 change ethanol sensitivity, alter sensitivity to an ethanol antagonist and can eliminate subunit-dependent differences in ethanol sensitivity between alpha1 and alpha2GlyRs. These findings suggest that the extracellular domain also represents a target for ethanol in GlyRs. We tested this hypothesis by investigating the effect of cysteine substitutions at positions 52 and 267 on responses to n-alcohols and propyl methanethiosulfonate (PMTS) in alpha1GlyRs. We also tested the role of Loop 2 in agonist activation and ethanol modulation by investigating the effect of cysteine point mutations at positions 50-60. Xenopus oocytes expressing human alpha1 wildtype (WT) or mutant GlyRs were voltage-clamped and tested 3-10 days post-injection. In support of the hypothesis, we found that: 1) The A52C mutation changed ethanol sensitivity compared to WT GlyRs