Bovine heart mitochondrial respiratory complex II generates ROS, mostly as superoxide, at the rate of about 20% of that detected during simultaneous operation of complex I and II when oxidation of ubiquinol is prevented by myxothiazol. ROS generating activity at different fumarate/succinate concentrations ratio implies that an enzyme component with a midpoint potential 40mV more positive than that of fumarate/succinate couple is the donor for one-electron reduction of oxygen. This suggests that the iron-sulfur cluster(s) is(are) involved in superoxide formation. Complex II-mediated ROS production exhibits a maximum at low (about 50μM) succinate concentration and gradually declines to zero activity upon further increase of the substrate. This phenomenology is explained and kinetically modeled to suggest a ping-pong mechanism of ROS generating activity where only dicarboxylate free reduced enzyme is oxidized by oxygen. The succinate:quinone reductase activity catalyzed by purified succinate:ubiquinone reductase also exhibits a ping-pong mechanism where only dicarboxylate free enzyme is oxidized by added quinone. Together these data suggest long distance interaction between the succinate (fumarate) binding and ubiquinone (ubiquinol) reactive sites.