Activation of mitochondrial ATP-sensitive potassium channels (mitoKATP channels) appears to protect the brain against ischaemic or chemical challenge (1). Unfortunately, the prototype mitoK_ATP channel opener, diazoxide, has potassium channel-independent pathways (2). We examined potential effects of BMS-191095, a novel, selective mitoK_ATP channel opener, against transient middle cerebral artery occlusion (MCAO) in rats (3). Experiments were performed on male Wistar rats subjected to 90 min of MCAO. Anesthesia was induced with 5% halothane in a 70:30 gas mixture of N₂O and O₂. Endotracheal intubation was performed, and all the animals were mechanically ventilated with 1.0-1.5% halothane in a 70:30 gas mixture of N₂O and O₂. BMS-191095 (25 μg; estimated final brain level of 40 μM, n=12) or vehicle was infused into the left lateral ventricle prior to the onset of ischaemia. We also examined effects of BMS-191095 on mitochondrial membrane potential and reactive oxygen species (ROS) production in cultured cortical neurons isolated from rat E18 fetuses. Mitochondrial membrane potential was monitored using the specific detection dye tetramethylrhodamine ethyl ester (TMRE, Molecular Probes). Treatment with BMS-191095 24 hr before the onset of ischaemia reduced total infarct volume by 29% (206.2 ± 16.5 to 149.2 ± 18.2 mm³; p<0.05) and cortical infarct volume by 34% reduction (144.2 ± 15.4 to 95.2 ± 14.4 mm³; p<0.05). However, treatment with BMS-191095 given 30 or 60 min prior to MCAO did not reduce infarct volume (p<0.05). Protective effects of BMS-191095 were completely prevented by co-treatment with 5-hydroxydecanoate (5-HD), a mitoK_ATP channel antagonist. In neurons, BMS-191095 (40 μM) depolarized the mitochondria without affecting levels of ROS, and this effect was inhibited by preincubation with 5-HD. In conclusion, BMS-191095 effectively protected the brain against MCAO in the rat, suggesting that selective opening of mitoK_ATP channels with BMS-191095 induces delayed preconditioning against transient focal ischaemia without non-specific effects such as generation of ROS.