The generation of reactive oxygen species by mitochondrial complex I (NADH:ubiquinone oxidoreductase) is considered a significant cause of cellular oxidative stress, linked to neuromuscular diseases and aging. Defining its mechanism is important for the formulation of causative connections between complex I defects and pathological effects. Oxygen is probably reduced at two sites in complex I, one associated with NADH oxidation in the mitochondrial matrix and the other associated with ubiquinone reduction in the membrane. Here, we study complex I from Escherichia coli, exploiting similarities and differences in the bacterial and mitochondrial enzymes to extend our knowledge of O2 reduction at the active site for NADH oxidation. E. coli and bovine complex I reduce O2 at essentially the same rate, with the same potential dependence (set by the NAD (+)/NADH ratio), showing that the rate-determining step is conserved. The potential dependent rate of H2O2 production does not correlate to the potential of the distal [2Fe-2S] cluster N1a in E. coli complex I, excluding it as the point of O2 reduction. Therefore, our results confirm previous proposals that O2 reacts with the fully reduced flavin mononucleotide. Assays for superoxide production by E. coli complex I were prone to artifacts, but dihydroethidium reduction showed that, upon reducing O2, it produces approximately 20% superoxide and 80% H2O2. In contrast, bovine complex I produces 95% superoxide. The results are consistent with (but do not prove) a specific role for cluster N1a in determining the outcome of O2 reduction; possible reaction mechanisms are discussed.