Despite its pharmacological relevance, the mechanism of the development of tolerance to the action of benzodiazepines is essentially unknown. The acute sedative action of diazepam is mediated via α1-GABAAreceptors. Therefore, we tested whether chronic activation of these receptors by diazepam is sufficient to induce tolerance to its sedative action. Knock-in mice, in which theα1-,α2-,α3-, orα5-GABAAreceptors had been rendered insensitive to diazepam by histidine-arginine point mutation, were chronically treated with diazepam (8 d; 15 mg · kg-1· d-1) and tested for motor activity. Wild-type, α2(H101R), and α3(H126R) mice showed a robust diminution of the motor-depressant drug action. In contrast, α5(H105R) mice failed to display any sedative tolerance. α1(H101R) mice showed no alteration of motor activity with chronic diazepam treatment. Autoradiography with [3H]flumazenil revealed no change in benzodiazepine binding sites. However, a decrease in α5-subunit radioligand binding was detected selectively in the dentate gyrus with specific ligands. This alteration was observed only in diazepam-tolerant animals, indicating that the manifestation of tolerance to the sedative action of diazepam is associated with a downregulation of α5-GABAAreceptors in the dentate gyrus. Thus, the chronic activation of α5-GABAAreceptors is crucial for the normal development of sedative tolerance to diazepam, which manifests itself in conjunction with α1-GABAAreceptors.