This work was focused on distinguishing the contribution of mitochondrial redox complexes to the production of reactive oxygen species (ROS) during cellular respiration. We were able to accurately measure, for the first time, the basal production of ROS under uncoupled conditions by using a very sensitive method, based on the fluorescent probe dichlorodihydrofluorescein diacetate. The method also enabled the detection of the ROS generated by the oxidation of the endogenous substrates in the mitochondrial preparations and could be applied to both mitochondria and live cells. Contrary to the commonly accepted view that complex III (ubiquinol:cytochrome c reductase) is the major contributor to mitochondrial ROS production, we found that complex I (NADH-ubiquinone reductase) and complex II (succinate-ubiquinone reductase) are the predominant generators of ROS during prolonged respiration under uncoupled conditions. Complex II, in particular, appears to contribute to the basal production of ROS in cells.