AbstractConsiderable evidence indicates that ethanol acts on specific residues in the transmembrane domains of glycine receptors (GlyRs). In this study, we tested the hypothesis that the extracellular domain is also a target for ethanol action by investigating the effect of cysteine substitutions at positions 52 (extracellular domain) and 267 (transmembrane domain) on responses to n‐alcohols and propyl methanethiosulfonate (PMTS) in α1GlyRs expressed in Xenopus oocytes. In support of the hypothesis: (i) The A52C mutation changed ethanol sensitivity compared to WT GlyRs; (ii) PMTS produced irreversible alcohol‐like potentiation in A52C GlyRs; and (iii) PMTS binding reduced the n‐chain alcohol cutoff in A52C GlyRs. Further studies used PMTS binding to cysteines at positions 52 or 267 to block ethanol action at one site in order to determine its effect at other site(s). In these situations, ethanol caused negative modulation when acting at position 52 and positive modulation when acting at position 267. Collectively, these findings parallel the evidence that established the TM domain as a target for ethanol, suggest that positions 52 and 267 are part of the same alcohol pocket and indicate that the net effect of ethanol on GlyR function reflects the summation of its positive and negative modulatory effects on different targets.