Although mitochondria are considered the most relevant site for the formation of reactive oxygen species (ROS) in cardiac myocytes, a major and unsolved issue is where ROS are generated in mitochondria. Respiratory chain is generally indicated as a main site for ROS formation. However, other mitochondrial components are likely to contribute to ROS generation. Recent reports highlight the relevance of monoamine oxidases (MAO) and p66(Shc). The importance of these systems in the irreversibility of ischemic heart injury will be discussed along with the cardioprotective effects elicited by both MAO inhibition and p66(Shc) knockout. Finally, recent evidence will be reviewed that highlight the relevance of mitochondrial ROS formation also in myocardial failure and atherosclerosis.