The aim of this study was to investigate the influence of reactive oxygen species (ROS) on the activity of complex I and on the cardiolipin content in bovine heart submitochondrial particles (SMP). ROS were generated through the use of xanthine/xanthine oxidase (X/XO) system. Treatment of SMP with X/XO resulted in a large production of superoxide anion, detected by acetylated cytochrome c method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to ROS generation resulted in a marked loss of complex I activity and to parallel loss of mitochondrial cardiolipin content. Both these effects were completely abolished by SOD+catalase. Exogenous added cardiolipin was able to almost completely restore the ROS-induced loss of complex I activity. No restoration was obtained with other major phospholipid components of the mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine, nor with peroxidized cardiolipin. These results demonstrate that ROS affect the mitochondrial complex I activity via oxidative damage of cardiolipin which is required for the functioning of this multisubunit enzyme complex. These results may prove useful in probing molecular mechanisms of ROS-induced peroxidative damage to mitochondria, which have been proposed to contribute to those pathophysiological conditions characterized by an increase in the basal production of reactive oxygen species such as aging, ischemia/reperfusion and chronic degenerative diseases.