Activation of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels protects the brain against ischemic or chemical challenge. Unfortunately, the prototype mitoK(ATP) channel opener, diazoxide, has mitoK(ATP) channel-independent actions. We examined the effects of BMS-191095, a novel selective mitoK(ATP) channel opener, on transient ischemia induced by middle cerebral artery occlusion (MCAO) in rats. Male Wister rats were subjected to 90 mins of MCAO. BMS-191095 (25 microg; estimated brain concentration of 40 micromol/L) or vehicle was infused intraventricularly before the onset of ischemia. In addition, the effects of BMS-191095 on plasma and mitochondrial membrane potentials and reactive oxygen species (ROS) production in cultured neurons were examined. Finally, we determined the effects of BMS-191095 on cerebral blood flow (CBF) and potassium currents in cerebrovascular myocytes. Treatment with BMS-191095 24 h before the onset of ischemia reduced total infarct volume by 32% and cortical infarct volume by 38%. However, BMS-191095 administered 30 or 60 mins before MCAO had no effect. The protective effects of BMS-191095 were prevented by co-treatment with 5-hydroxydecanoate (5-HD), a mitoK(ATP) channel antagonist. In cultured neurons, BMS-191095 (40 micromol/L) depolarized the mitochondria without affecting ROS levels, and this effect was inhibited by 5-HD. BMS-191095, similar to the vehicle, caused an unexplained but modest reduction in the CBF. Importantly, BMS-191095 did not affect either the potassium currents in cerebrovascular myocytes or the plasma membrane potential of neurons. Thus, BMS-191095 afforded protection against cerebral ischemia by delayed preconditioning via selective opening of mitoK(ATP) channels and without ROS generation.