Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure.