Fluoxetine has been reported to be a novel allosteric modulator of GABA(A) receptors with the notable exception of receptors that contain the alpha5-subunit isoform [Robinson, R.T., Drafts, B.C., Fisher, J.L., 2003. Fluoxetine increases GABA(A) receptor activity through a novel modulatory site. J. Pharmacol. Exp. Ther. 304, 978-984]. A mutagenic strategy has been used to investigate the structural basis for the insensitivity of this subunit. An alpha1/alpha5-subunit chimeragenesis approach first demonstrated the importance of the alpha1-subunit N-terminal sequence E165-D183 (corresponding to alpha5 E169-D187) in fluoxetine modulation. Specific amino acid substitutions in this domain subsequently revealed that a single mutation in the alpha5-subunit to the equivalent residue in alpha1 (T179A) was sufficient to confer fluoxetine sensitivity to the alpha5-containing receptor. However, the reciprocal mutation in the alpha1-subunit (A175T) did not result in a loss in sensitivity, suggesting the involvement of additional determinants for fluoxetine modulation. A comparative modeling approach was used to probe amino acids that may lie in close proximity to alpha1A175. This led serendipitously to the identification of a specific residue, alpha1F45, which, when mutated to an alanine, resulted in a significant decrease in potency for activation of the receptor by GABA and also reduced the efficacies of the partial agonists, THIP and P4S.