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Gamma-aminobutyric acid, type A (GABAA) receptors are complex heterogeneous pentamers with various drug binding sites. Several lines of evidence suggest that benzodiazepines modulate certain GABAA receptors in a flumazenil-insensitive manner, possibly via binding sites other than the classical ones. However, GABAA receptor subtypes that contain non-classical benzodiazepine binding sites are not systemically studied. The present study investigated the high-concentration effects of three benzodiazepines and their sensitivity to flumazenil on different recombinant (α1β2, α2β2, α3β2, α4β2, α5β2 and α1β3) and native neuronal GABAA receptors using the whole-cell patch-clamp electrophysiology technique. The classical benzodiazepine diazepam (200 μmol/L) and midazolam (200 μmol/L) produced flumazenil-insensitive effects on α1β2 receptor, whereas the imidazopyridine zolpidem failed to modulate the receptor. Flumazenil-insensitive effects of diazepam were also observed on the α2β2, α3β2 and α5β2, but not α4β2 receptors. Unlike β2-containing receptors, the α1β3 receptor was insensitive to diazepam. Moreover, the diazepam (200 μmol/L) effects on some cortical neurons could not be fully antagonized by flumazenil (200 μmol/L). These findings suggested that the non-classical (flumazenil-insensitive) benzodiazepine effects depended on certain receptor subtypes and benzodiazepine structures and may be important for designing of subtype- or binding site- specific drugs.