The present investigation deals with the suggested role of redox-cycling ubisemiquinones in mitochondrial O2-. generation. Due to the functional complexity of electron-transferring ubiquinones in the respiratory chain, model experiments were designed to study whether ubisemiquinones will directly react with oxygen, thereby generating O2-. radicals. Based on the fact that mitochondrial ubiquinone was reported to operate in an aprotic surrounding of the inner mitochondrial membrane, the reactivity of ubisemiquinones with oxygen was tested in water-free acetonitrile. Our results prove that autoxidation of ubisemiquinones requires the addition of protons to the non-polar reaction system. An experimental evaluation of the validity of this finding with respect to mitochondrial ubiquinones is impeded by the biochemical role that oxygen plays in the establishment of ubisemiquinone populations. To differentiate between a possible direct interaction of oxygen on redox-cycling ubisemiquinones and this indirect biochemical O2 effect, we have successfully introduced ferricyanide instead of oxygen to establish mitochondrial ubisemiquinone pools. Ubisemiquinones in this reaction system were not susceptible to oxygen and no O2-. radicals were released unless the inner mitochondrial membrane was protonated by toluene pretreatment. Since the inner mitochondrial membrane is normally not permeable to protons (which is a prerequisite of the chemiosmotic theory of energy conservation) based on our experiments we can exclude the involvement of redox-cycling ubisemiquinones in mitochondrial O2-. generation.