Manganese-induced toxicity has been linked to mitochondrial dysfunction and an increased generation of reactive oxygen species (ROS). We could recently show in mechanistic studies that Mn 2+ ions induce hydrogen peroxide (H 2 O 2 ) production from the ubiquinone binding site of mitochondrial complex II (II Q ) and generally enhance H 2 O 2 formation by accelerating the rate of superoxide dismutation. The present study with intact mitochondria reveals that manganese additionally enhances H 2 O 2 emission by inducing mitochondrial permeability transition (mPT). In mitochondria fed by NADH-generating substrates, the combination of Mn 2+ and different respiratory chain inhibitors led to a dynamically increasing H 2 O 2 emission which was sensitive to the mPT inhibitor cyclosporine A (CsA) as well as Ru-360, an inhibitor of the mitochondrial calcium uniporter (MCU). Under these conditions, flavin-containing enzymes of the mitochondrial matrix, e.g. the mitochondrial 2-oxoglutaratedehydrogenase (OGDH), were major sources of ROS. With succinate as substrate, Mn 2+ stimulated ROS production mainly at complex II, whereby the applied succinate concentration had a marked effect on the tendency for mPT. Also Ca 2+ increased the rate of H 2 O 2 emission by mPT, while no direct effect on ROS-production of complex II was observed. The present study reveals a complex scenario through which manganese affects mitochondrial H 2 O 2 emission: stimulating its production from distinct sites (e.g. site II Q ), accelerating superoxide dismutation and enhancing the emission via mPT which also leads to the loss of soluble components of the mitochondrial antioxidant systems and favors the ROS production from flavin-containing oxidoreductases of the Krebs cycle. [ABSTRACT FROM AUTHOR]