Diazoxide opening of the mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP) protects the heart against ischemia-reperfusion injury by unknown mechanisms. We investigated the mechanisms by which mitoK_ATP opening may act as an end effector of cardioprotection in the perfused rat heart model, in permeabilized fibers, and in rat heart mitochondria. We show that diazoxide pretreatment preserves the normal low outer membrane permeability to nucleotides and cytochrome c and that these beneficial effects are abolished by the mitoK_ATP inhibitor, 5-hydroxydecanoate. We hypothesize that an open mitoK_ATP during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP. This hypothesis is supported by findings in mitochondria showing that small decreases in intermembrane space volume, induced either by osmotic swelling or by diazoxide, increased the K_1/2 for ADP stimulation of respiration and sharply reduced ATP hydrolysis. These effects are proposed to lead to preservation of adenine nucleotides during ischemia and efficient energy transfer upon reperfusion.