The aim of this study was to further elucidate the mechanisms involved in adaptive changes of GABA A receptors following prolonged exposure to flumazenil, the antagonist of benzodiazepine binding sites on GABA A receptors. The effects of prolonged flumazenil treatment were studied on recombinant α 1 β 2 γ 2S GABA A receptors stably expressed in human embryonic kidney (HEK 293) cells. Using radioligand binding experiments we found enhancement in the maximum number of [3H]muscimol labeled binding sites in different preparations of HEK 293 cells. The parallel increase of [3H]flunitrazepam binding sites in the membranes was reduced in the presence of actinomycin D and cycloheximide, inhibitors of RNA and protein synthesis, respectively. Chronic flumazenil also raised the steady-state level of mRNA encoding α 1 receptor subunit. The results suggest that the up-regulation of GABA A receptors, observed after prolonged flumazenil treatment is at least partly due to increased de novo synthesis of receptor proteins at both transcriptional and translational level.