This paper is published as Journal Series No. 9794, Agricultural Research Division, University of Nebraska.Ferric leghemoglobin reductase (FLbR) from soybean (Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb+3), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O2 was removed from the reaction system, but all were restored upon readdition of O2. In the absence of exogenous electron carriers and in the presence of O2 and excess NADH, FLbR catalyzed NADH oxidation with the generation of H2O2 functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 µg/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb+3 reduction. The formation of H2O2 during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H2O2. The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb+3 reduction were not constant but changed with time and with concentrations of NADH and O2 in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb+3 by reaction intermediates. A study of the affinity of FLbR for O2 showed that the enzyme required at least micromolar levels of dissolved O2 for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems.The work was supported by the National Research Initiative Competitive Grants Program/U.S. Department of Agriculture (grant No. 91-37305-6705) to R.V.K. and by the Consejo Asesor de Investigación-Diputación General de Aragón (grant No. PCB-10/90) to M.B.Peer reviewed