The present studies used increased atmospheric pressure in place of a traditional pharmacological antagonist to probe the molecular sites and mechanisms of ethanol action in glycine receptors (GlyRs). Based on previous studies, we tested the hypothesis that physical-chemical properties at position 52 in extracellular domain Loop 2 of alpha1GlyRs, or the homologous alpha2GlyR position 59, determine sensitivity to ethanol and pressure antagonism of ethanol. Pressure antagonized ethanol in alpha1GlyRs that contain a non-polar residue at position 52, but did not antagonize ethanol in receptors with a polar residue at this position. Ethanol sensitivity in receptors with polar substitutions at position 52 was significantly lower than GlyRs with non-polar residues at this position. The alpha2T59A mutation switched sensitivity to ethanol and pressure antagonism in the WTalpha2GlyR, thereby making it alpha1-like. Collectively, these findings indicate that (i) polarity at position 52 plays a key role in determining sensitivity to ethanol and pressure antagonism of ethanol; (ii) the extracellular domain in alpha1- and alpha2GlyRs is a target for ethanol action and antagonism and (iii) there is structural-functional homology across subunits in Loop 2 of GlyRs with respect to their roles in determining sensitivity to ethanol and pressure antagonism of ethanol. These findings should help in the development of pharmacological agents that antagonize ethanol.