Hydrogenases control the H-2-related metabolism in many microbes. Most of these enzymes are prone to immediate inactivation by O-2. However, a few members of the subclass of [NiFe]-hydrogenases are able to convert H-2 into protons and electrons even in the presence of O-2, making them attractive for biotechnological application. Recent studies on O-2-tolerant membrane-bound hydrogenases indicate that the mechanism of O-2 tolerance relies on their capability to completely reduce O-2 with four electrons to harmless water. In order to verify this hypothesis, we probed the O-2 reduction capacity of the soluble, NAD(+)-treducing [NiFe]-hydrogenase (SH) from Ralstonia eutropha H16. A newly established, homologous overexpression allowed the purification of up to 90 mg of homogeneous and highly active enzyme from 10 g of cell material. We showed that the SH produces trace amounts of superoxide in the course of H-2-driven NAD+ reduction in the presence of O-2. However, the major products of the SH-mediated oxidase activity was in fact hydrogen peroxide and water as shown by the mass spectrometric detection of (H2O)-O-18 formed from H-2 and isotopically labeled O-18(2). Water release was also observed when the enzyme was incubated with NADH and O-18(2), demonstrating the importance of reverse electron flow to the [NiFe] active site for O-2 reduction. A comparison of the turnover rates for H-2 and O-2 revealed that in the presence of twice the ambient level of O-2, up to 3% of the electrons generated through H-2 oxidation serve as "health insurance" and are reused for O-2 reduction.