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Benzodiazepines (BZDs) produce versatile pharmacological actions through positive modulation of GABA_A receptors (GABA_ARs). A previous study has demonstrated that high concentrations of diazepam potentiate GABA currents on the α₁β₂γ₂ and α₁β₂ GABA_ARs in a flumazenil-insensitive manner. In this study, the high-concentration effects of BZDs and their sensitivity to flumazenil were determined on synaptic (α₁β₂γ₂, α₂β₂γ₂, α₅β₂γ₂) and extra-synaptic (α₄β₂δ) GABA_ARs using the voltage-clamp electrophysiology technique. The in vivo evaluation of flumazenil-insensitive BZD effects was conducted in mice via the loss of righting reflex (LORR) test. Diazepam induced biphasic potentiation on the α₁β₂γ₂, α₂β₂γ₂ and α₅β₂γ₂ GABA_ARs, but did not affect the α₄β₂δ receptor. In contrast to the nanomolar component of potentiation, the second potentiation elicited by micromolar diazepam was insensitive to flumazenil. Midazolam, clonazepam, and lorazepam at 200 µM exhibited similar flumazenil-insensitive effects on the α₁β₂γ₂, α₂β₂γ₂ and α₅β₂γ₂ receptors, whereas the potentiation induced by 200 µM zolpidem or triazolam was abolished by flumazenil. Both the GABA_AR antagonist pentylenetetrazol and Fa173, a proposed transmembrane site antagonist, abolished the potentiation induced by 200 µM diazepam. Consistent with the in vitro results, flumazenil antagonized the zolpidem-induced LORR, but not that induced by diazepam or midazolam. Pentylenetetrazol and Fa173 antagonized the diazepam-induced LORR. These findings support the existence of non-classical BZD binding sites on certain GABA_AR subtypes and indicate that the flumazenil-insensitive effects depend on the chemical structures of BZD ligands.