Mitochondrial respiration produces both complete and partially reduced oxygen species that are involved in physiological and pathological processes. Indeed, unspecific oxidative damage induced by excessive mitochondrial reactive oxygen species (ROS) plays a role in aging and several diseases, whereas low amounts of ROS act in physiological signaling processes. The exact molecular species, the rate, and the conditions of mitochondrial ROS release are not clearly evaluable by current methods based on oxidation sensitive markers. Recently, electrochemical analysis of biological samples has improved. Following latest methodology, we implemented a novel electrochemical assay for the investigation of aerobic metabolism in isolated mitochondria through simultaneous measurement of O2 consumption and H2O 2 production. Our experiments confirm active H2O 2 production by respiring mouse liver mitochondria and show that ATP synthase activation increases the rate of H2O2, suggesting that state 3 mitochondria might induce the cell through oxidative signals.