The study investigates the mechanisms of superoxide production and release in mitochondria, focusing on Complex I and Complex III. Complex I-dependent superoxide is found to be exclusively released into the mitochondrial matrix, while no detectable levels escape from intact mitochondria. In contrast, Complex III releases superoxide to both the mitochondrial matrix and the intermembrane space. The authors propose two models to explain how superoxide from Complex III can reach both sides of the inner mitochondrial membrane: one involving the diffusion of neutral semiquinone through a hydrophobic tunnel and protonation at the lipid-aqueous phase interface, and the other involving proton-coupled electron transfer in the hydrophobic part of the semiquinone. These findings challenge the prevailing view that Complex III only releases superoxide to the cytoplasmic side of the inner mitochondrial membrane.The study investigates the mechanisms of superoxide production and release in mitochondria, focusing on Complex I and Complex III. Complex I-dependent superoxide is found to be exclusively released into the mitochondrial matrix, while no detectable levels escape from intact mitochondria. In contrast, Complex III releases superoxide to both the mitochondrial matrix and the intermembrane space. The authors propose two models to explain how superoxide from Complex III can reach both sides of the inner mitochondrial membrane: one involving the diffusion of neutral semiquinone through a hydrophobic tunnel and protonation at the lipid-aqueous phase interface, and the other involving proton-coupled electron transfer in the hydrophobic part of the semiquinone. These findings challenge the prevailing view that Complex III only releases superoxide to the cytoplasmic side of the inner mitochondrial membrane.