L-655,708 is a ligand for the benzodiazepine site of the γ-aminobutyric acid type A (GABAA) receptor that exhibits a 100-fold higher affinity for α5-containing receptors compared with α1-containing receptors. Molecular biology approaches have been used to determine which residues in the α5 subunit are responsible for this selectivity. Two amino acids have been identified, α5Thr208 and α5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at α5-containing receptors. L-655,708 is a partial inverse agonist at the GABAA receptor which exhibited no functional selectivity between α1- and α5-containing receptors and showed no change in efficacy at receptors containing α1 subunits where amino acids at both of the sites had been altered to their α5 counterparts (α1ΔSer205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABAA receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor α subunit that contributes to this binding site.