Abstract The study aimed to determine the effect of 3?,4?-dihydroxyflavonol (DiOHF) on mitochondrial function, in particular opening of the mitochondrial permeability transition pore (mPTP), respiratory function and reactive oxygen species (ROS) production, in isolated cardiac mitochondria after coronary artery occlusion and reperfusion in vivo. Opening of the mPTP, oxygen consumption and ROS production (assessed by measurement of H2O2) was determined in mitochondria isolated from normal hearts or from the ischemic zone of rat hearts subjected to 30 min coronary artery occlusion and 15 min reperfusion. Treatment of sham rats with DiOHF (10 mg kg?1 iv) significantly increased the concentration of Ca2+ required to stimulate mPTP opening. This was accompanied by increased state 3 oxygen consumption and decreased H2O2 release. Ischemia and reperfusion (IR) significantly decreased the concentration of Ca2+ required to stimulate mPTP opening, decreased state 3 oxygen consumption and increased H2O2 release, when pyruvate plus malate was provided as a substrate. Treatment with DiOHF prevented IR-induced changes in mPTP opening, state 3 oxygen consumption and H2O2 release so that there was no difference compared to sham. In isolated cardiac mitochondria from normal rats DiOHF had no effect on mPTP opening or on state 3 respiration but caused a small increase in state 4 respiration and decreased the respiratory control ratio.