The mitochondrial respiratory chain is a major source of cellular reactive oxygen species (ROS). Recent data indicate that beside the established H2O2/O2●–-generators NADH-ubiquinone- (complex I) and the ubiquinone-cytrochrome c-oxidoreductase (complex III), additional sources significantly contribute to the overall ROS production. These include the oxoglutarate dehydrogenase complex and the succinate dehydrogenase (complex II; SDH). The latter was shown to produce mainly H2O2 from its FAD site. More recently manganese toxicity in the central nervous system has been linked to an elevated ROS production by complex II. However, the molecular mechanism as well as the distinct site of origin remained elusive. In this study with submitochondrial particles (SMP) and isolated rat heart mitochondria (RHM) we confirmed an inducing effect of manganese on ROS production by complex II. Furthermore, we tracked down H2O2/O2●–-to be derived mainly from the ubiquinone binding site. In RHM, manganese additionally acted as a potent inducer of mitochondrial permeability transition, and hence potentiated the emission of H2O2 from ‘substrate’ oxidoreductases of the Krebs cycle. Moreover, manganese generally led to an enhanced emission of hydrogen peroxide due to its accelerating effect on superoxide dismutation. These findings highlight the complex effects of manganese on mitochondrial H2O2 emission. They also warrant further explorations aiming to understand/predict the functional consequences of H2O2/O2●–formed at distinct sites of SDH.