The extracellular α (+)/γ 2 (-) interface in the α 1,2,3,5βγ 2 GABA A receptor harbours the allosteric binding site targeted by benzodiazepines and newer generations of subtype-selective modulators. We have probed the molecular determinants for the affinity/potency-based α 1-preference exhibited by the hypnotic zolpidem (Ambien®, Stilnox®) and the efficacy-based α 3-over-α 1 selectivity displayed by the analgesic NS11394. Binding affinities and functional properties of the modulators were characterized at wild-type, concatenated, mutant and chimeric α 1,3β 2γ 2S receptors expressed in tsA201 cells and Xenopus oocytes by [ 3H]flumazenil binding and two-electrode voltage clamp electrophysiology. Substitution of Gly 201 in α 1 with the corresponding Glu in α 3 completely eliminated the α 1-over-α 3 preference exhibited by zolpidem. In contrast, the reverse α 3-E225G mutation did not yield corresponding increases in the binding affinity or modulatory potency of zolpidem at α 3β 2γ 2S, and two additional molecular elements in the extracellular domain of the α-subunit were found also to contribute to its α 1-preference. Interestingly, the α 1-Gly 201/α 3-Glu 225 residue was also a key determinant of the efficacy-based α 3-over-α 1 selectivity exhibited by NS11394, and a pronounced correlation existed between the side-chain bulkiness of this residue and the modulatory efficacy of NS11394 at the receptor. The subtype-selectivity determinants identified for zolpidem and NS11394 were found also to apply in different degrees to the α 1-preferring modulator indiplon and the α 3-over-α 1 selective modulator L-838,417, respectively. In conclusion, the molecular origins of subtype-selectivity exhibited by benzodiazepine-site modulators at the α 1,2,3,5βγ 2 GABA A receptor seem more complex than previously appreciated, and the importance of the α 1-Gly 201/α 3-Glu 225 residue for both potency- and efficacy-based subtype-selective modulation through this site is likely to be rooted in different molecular mechanisms.