H2O-forming NADH oxidase from Leuconostoc mesenteroides subsp. mesenteroides NRIC 1541(T) and H2O2-forming NADH oxidase from Sporolactobacillus inulinus NRIC 1133(T) were purified to electrophoretic homogeneity. These enzymes exhibited the absorption spectrum typical of a flavoprotein, and their activities were inhibited by inhibitors of the flavoprotein enzyme such as quinine and quinacrine. In addition, cofactors of the above-mentioned enzymes were identified as FAD. L. mesenteroides subsp. mesenteroides NADH oxidase catalyzed the reduction of oxygen to water with beta-NADH as the substrate, while S. inulinus NADH oxidase catalyzed the reduction of oxygen to hydrogen peroxide. In addition, L. mesenteroides subsp. mesenteroides NADH oxidase activity was slightly enhanced by the addition of FAD to the reaction mixture whereas S. inulinus NADH oxidase activity was markedly enhanced. In the absence of free FAD, neither enzyme catalyzed electron transfer from beta-NADH to hydrogen peroxide. In the presence of free FAD, S. inulinus NADH oxidase catalyzed electron transfer from beta-NADH to hydrogen peroxide, but L. mesenteroides subsp. mesenteroides NADH oxidase did not. This finding suggests that S. inulinus NADH oxidase has an NADH peroxidase activity. L. mesenteroides subsp. mesenteroides contains NADH oxidase and NADH peroxidase, and an NADH oxidase/NADH peroxidase coupling system is thought to control the intracellular redox balance and protect this bacterium from hydrogen peroxide toxicity. By contrast, a single enzyme in S. inulinus exhibits both NADH oxidase and NADH peroxidase activity. Consequently, despite the fact that they lack the respiratory chain and catalase, these bacteria grow well under aerobic conditions. The N-terminal amino acid sequence of L. mesenteroides subsp. mesenteroides NADH oxidase is very similar to those of other H2O-forming NADH oxidases. On the other hand, the N-terminal amino acid sequence of S. inulinus NADH oxidase is very similar to those of other H2O2-forming NADH oxidases.