The mechanism of mitochondrial damage during reperfusion injury of ischemic myocardium was studied using mongrel dogs in vivo and isolated mitochondria in vitro. Seventy-seven adult dogs were divided into three groups: the control group (n = 38), the Coenzyme Q10 (CoQ10)-5 mg group (n = 24), and the CoQ10-15 mg group (n = 15). In the control group, the left anterior descending coronary artery (LAD) of the dog was occluded for 15 min followed by 5 min of reperfusion after 40 min of premedication with physiological saline. In both CoQ10 groups, 5 mg/kg or 15 mg/kg of CoQ10 was infused intravenously for 20 min and then physiological saline was administered for 20 min before 15 min occlusion of the LAD. Subsequently, reperfusion was allowed for 5 min. Each group was further divided into two subgroups depending on the presence (arrhythmia group) or the absence (non-arrhythmia group) of ventricular arrhythmias. Immediately after 15 min occlusion, myocardial samples were taken from the normal and reperfused areas to measure CoQ10 content of myocardium. Heart mitochondria were prepared after 5 min of reperfusion from both areas. Arrhythmias appeared in 12 of 38 dogs in the control group (32%), two of 24 dogs in the CoQ10-5 mg group (8%) and none of 15 dogs in the CoQ10-15 mg group (0%). Premedication with CoQ10 increased tissue CoQ10 content in a dose-dependent manner. In the CoQ10-5 mg group, the increase in CoQ10 content of dogs with reperfusion arrhythmias was relatively less than that of dogs without reperfusion arrhythmias. In each group, mitochondrial function was decreased in the arrhythmia group compared to that of the non-arrhythmia group. The increase in free fatty acid (FFA) content and the decrease in phospholipid content were also observed in mitochondria from the reperfused area of each arrhythmia group. The increase in FFA and mitochondrial dysfunction were induced by the incubation of mitochondria in vitro with phospholipase (PLase) A2 or PLase C, and protected by the addition of CoQ10. These results suggest that PLase plays an important role in the development of mitochondrial damage associated with reperfusion.