The regeneration of NAD was carried out with an NADH oxidase from the gram-positive bacterium Lactobacillus brevis DSM 20 054. High levels of this enzyme are produced by L. brevis during aerobic growth. The complete nucleotide sequence of the NADH oxidase gene was determined and the primary structure of the enzyme was deduced. The gene shows an open reading frame coding for 450 amino acids. The protein has a calculated pI of 4.76 and theoretical molecular mass of 48.9 kDa, which agrees well with the molecular mass of 50 kDa for the subunit as determined by SDS-PAGE. The GC-content of the nox gene (46.6%) is consistent with the general GC-content of L. brevis (46.4%). The primary structure of this enzyme shows homologies with other bacterial NADH oxidase. According to the PFAM database of protein domains the NADH oxidase from L brevis belongs to the family of FAD-dependent pyridine nucleotide reductases (FADPNR). The application of this NADH oxidase for the regeneration of NAD is demonstrated by the simultaneous coupling with an NAD-dependent R-specific alcohol dehydrogenase. Starting with R,S-1-phenylethanol, the R-enantiomer could be oxidized completely resulting in a pure S-alcohol.