Amino acids in the alpha- and gamma-subunits contribute to the benzodiazepine binding site of GABA(A)-receptors. We show that the mutation of a conserved histidine residue in the N-terminal extracellular segment (alpha1H101R, alpha2H101R, alpha3H126R, and alpha5H105R) results not only in diazepam-insensitivity of the respective alphaxbeta2,3gamma2-receptors but also in an increased potentiation of the GABA-induced currents by the partial agonist bretazenil. Furthermore, Ro 15-4513, an inverse agonist at wild-type receptors, acts as an agonist at all mutant receptors. This conserved molecular switch can be exploited to identify the pharmacological significance of specific GABA(A)-receptor subtypes in vivo.