The major isoform of the GABAA receptor is α1β2γ2. The binding sites for the agonist GABA are located at the β2+/α1- subunit interfaces and the modulatory site for benzodiazepines at α1+/γ2-. In the absence of α1 subunits, a receptor was formed that was gated by GABA and modulated by diazepam similarly. This indicates that alternative subunits can take over the role of the α1 subunits. Point mutations were introduced in β2 or γ2 subunits at positions homologous to α1- benzodiazepine binding and GABA binding positions, respectively. From this mutation work we conclude that the site for GABA is located at a β2+/β2- subunit interface and that the diazepam site is located at the β2+/γ2- subunit interface. Computational docking leads to a structural hypothesis attributing this non-canonical interaction to a binding mode nearly identical with the one at the α1+/γ2- interface. Thus, the β2 subunit can take over the role of the α1 subunit for the formation of both sites, its minus side for the GABA binding site and its plus side for the diazepam binding site.