The Gram-positive bacterium Rhodococcus opacus DSM 43250 produces an L-amino acid oxidase (L-AAO) with a very broad substrate specificity. This enzyme has been purified to homogeneity and a detailed biochemical characterization was carried out. The complete nucleotide sequence of the L-AAO gene was determined and the primary structure Of L-AAO was deduced. The molecular mass of the native enzyme was 99 kDa determined by gel filtration, 54.2/108.5 kDa measured by MALDI-TOF/MS, 53.2 kDa for the subunit calculated after SDS/PAGE. The coenzyme-binding motif G-X-G-X-X-G which is known for all L-AAOs was found very close to the N-terminus of the protein. L-AAO oxidized 39 out of 43 tested L-amino acids. The kinetic data for 16 of these L-ammo acids were determined revealing K-m-values in the range of 15-30 muM for substrates like L-phenylalanine, L-leucine, L-citrulline and L-lysine. The stability Of L-AAO can be increased by storage or incubation of the enzyme in glycine/NaOH buffer. The protein has a pI of 4.8 and a slightly basic pH-optimum at pH 8-9 measured for L-alanine, L-phenylalanine and L-leucine as substrates. The ability for resolution of racemic mixtures was investigated and D-amino acids with an enantiomeric excess of >99% were obtained.