Diazepam is used clinically for its myorelaxant, anxiolytic, sedative, and anticonvulsant properties. Although the anxiolytic action is mediated by α2 γ-aminobutyric acid A (GABA A ) receptors, the sedative action and in part the anticonvulsant action are mediated by α1 GABA A receptors. To identify the GABA A receptor subtypes mediating the action of diazepam on muscle tone, we have assessed the myorelaxant properties of diazepam in α2(H101R) and α3(H126R) knock-in mice harboring diazepam-insensitive α2 or α3 GABA A receptors, respectively. Whereas in α2(H101R) mice the myorelaxant action of diazepam was almost completely abolished at doses up to 10 mg/kg, the same dose induced myorelaxation in both wild-type and α3(H126R) mice. It was only at a very high dose (30 mg/kg diazepam) that α2(H101R) mice showed partial myorelaxation and α3(H126R) mice were partially protected from myorelaxation compared with wild-type mice. Thus, the myorelaxant activity of diazepam seems to be mediated primarily by α2 GABA A receptors and at high concentrations also by α3 GABA A receptors.