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Mechanisms of mitochondrial superoxide formation remain poorly understood despite considerable medical interest in oxidative stress. Superoxide is produced from both Complexes I and III of the electron transport chain, and once in its anionic form it is too strongly charged to readily cross the inner mitochondrial membrane. Thus, superoxide production exhibits a distinct membrane sidedness or "topology." In the present work, using measurements of hydrogen peroxide (Amplex red) as well as superoxide (modified Cypridina luciferin analog and aconitase), we demonstrate that Complex I-dependent superoxide is exclusively released into the matrix and that no detectable levels escape from intact mitochondria. This finding fits well with the proposed site of electron leak at Complex I, namely the iron-sulfur clusters of the (matrix-protruding) hydrophilic arm. Our data on Complex III show direct extramitochondrial …