Complex III releases superoxide to both sides of the inner mitochondrial membrane. Despite previous studies suggesting that superoxide is mainly released into the matrix, this study shows that Complex III can also release superoxide to the intermembrane space. Using measurements of hydrogen peroxide and superoxide, the researchers found that Complex I-dependent superoxide is exclusively released into the matrix, while Complex III-derived superoxide can be released to both sides. The study also shows that the remaining 50% of electron leak from Complex III must be due to superoxide released into the matrix. The data confirm that Complex III can release superoxide to both sides of the inner mitochondrial membrane. The study also discusses two models explaining how superoxide can reach both sides of the inner membrane. The first model suggests that a neutral ubisemiquinone can diffuse out of the Qo site and react with oxygen to form aqueous superoxide. The second model suggests that superoxide is formed in the hydrophobic part of the Qo site via a proton-coupled electron transfer. The results of this study support the hypothesis that Complex III can release superoxide to both sides of the inner mitochondrial membrane.Complex III releases superoxide to both sides of the inner mitochondrial membrane. Despite previous studies suggesting that superoxide is mainly released into the matrix, this study shows that Complex III can also release superoxide to the intermembrane space. Using measurements of hydrogen peroxide and superoxide, the researchers found that Complex I-dependent superoxide is exclusively released into the matrix, while Complex III-derived superoxide can be released to both sides. The study also shows that the remaining 50% of electron leak from Complex III must be due to superoxide released into the matrix. The data confirm that Complex III can release superoxide to both sides of the inner mitochondrial membrane. The study also discusses two models explaining how superoxide can reach both sides of the inner membrane. The first model suggests that a neutral ubisemiquinone can diffuse out of the Qo site and react with oxygen to form aqueous superoxide. The second model suggests that superoxide is formed in the hydrophobic part of the Qo site via a proton-coupled electron transfer. The results of this study support the hypothesis that Complex III can release superoxide to both sides of the inner mitochondrial membrane.