Abstract gamma-Aminobutyric acid type A (GABA(A)) receptors are molecular targets for alcohols. Previous work suggests that S270 and A291 residues in the transmembrane (TM) 2 and 3 domains of the GABA(A) receptor alpha subunit are components of an alcohol-binding pocket, and S270I and A291W mutants abolished ethanol potentiation. Our results showed that A295C and F296C residues in the TM3 of the GABA(A) receptor alpha1 subunit are accessible to hexylmethanethiosulfonate (HMTS) in the alcohol-bound state, but not in the resting state. Thus, the A295C and F296C sites become water-accessible as a result of alcohol-induced conformational changes. If S270 or A291 residues are sites of alcohol binding, then S270I or A291W mutations should prevent alcohol-induced conformational movements within the TM3 domain. To investigate this question, the accessibility of HMTS reagent to double mutants (A291W/A295C, A291W/F296C, S270I/A295C or S270I/F296C) in the presence of ethanol or hexanol was tested. The A291W or S270I mutations markedly reduced the accessibility of HMTS to all the double mutants in the ethanol-bound state, and to S270I/F296C, A291W/A295C and A291W/F296C double mutants in the hexanol-bound state, suggesting that the A291 or S270 residues are critical sites for alcohol binding and alcohol-induced conformational changes.