Diazoxide is the prototypical opener of mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) and protects neurons in vivo and in vitro against chemical and anoxic stresses. While we have previously shown that diazoxide administration induces acute preconditioning against transient cerebral ischemia in rats, the potential for delayed preconditioning of diazoxide has not been examined. The purpose of this study was to determine whether diazoxide promotes delayed preconditioning following 90 min of middle cerebral artery occlusion (MCAO) in male Wistar rats. Diazoxide (10 mg/kg) or vehicle was injected intraperitoneally 24 h before MCAO. Infarct volumes were measured 72 h after reperfusion. In animals anesthetized with halothane, treatment with diazoxide exhibited a 35% reduction (48.3+/-3.0% to 31.3+/-4.8%) and 18% reduction (35.1+/-2.2% to 28.9+/-2.1%) in cortical and subcortical infarct volumes, respectively. Administration of the mitoK(ATP) blocker 5-hydroxydecanoate attenuated this beneficial effect. In contrast, diazoxide did not induce delayed preconditioning in isoflurane-anesthetized rats. These findings support the concept that diazoxide produces delayed preconditioning via mitoK(ATP) activation but that physiological status can affect induction of preconditioning.