GABA_A receptors mediate most of the fast inhibitory neurotransmission in the brain. Prolonged occupancy of these receptors by ligands leads to regulatory changes often resulting in reduction of receptor function. The mechanism of these changes is still unknown. In this study, stably transfected human embryonic kidney (HEK) 293 cells were used as a model to study the effects of prolonged flumazenil (antagonist of benzodiazepine binding sites at GABA_A receptors) exposure on the recombinant ₁β₂γ_2S GABA_A receptors, the most common type of GABA_A receptors found in the brain. Exposure (48 h) of HEK 293 cells stably expressing recombinant ₁β₂γ_2S GABA_A receptors to flumazenil (1 or 5 μM) in the presence of GABA (1 μM), enhanced the maximum number (B_max) without affecting the affinity (K_d) of [³H]TBOB labeled binding sites for convulsants. Diazepam (1 nM–1 mM) in the presence of GABA (1 μM) modulated [³H]TBOB binding to control and flumazenil pretreated cells according to a two-site model. No significant differences between the groups were observed in either the potency or efficacy of diazepam to modulate [³H]TBOB binding, as evidenced by a lack of significant changes between their IC₅₀ and I_max values. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant ₁β₂γ_2S GABA_A receptors to flumazenil up-regulates the binding sites for convulsants, but it does not appear to affect the functional coupling between these sites and benzodiazepine binding sites. Along with our recent data, these results suggest that chronic treatment with flumazenil enhances the number of GABA_A receptors.