The L-amino acid oxidase (L-aao) from Bacillus carotarum 2Pfa was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from crude sonicated cell extract by a combination of anion exchange chromatography and gel filtration. The purified enzyme was a dimer with a native relative molecular mass of approximately 102,000 to 115,000 and comprised two identical subunits of 54,000. The isoelectric point of the L-aao was at pH 4.8, the pH optimum was at 8.0-8.5 and the temperature optimum was at approximately 50 degrees C. It was stable for several months at +4 degrees C and at -20 degrees C. The enzyme contained 2 mol flavin adenine dinucleotide (FAD)/mol enzyme and exhibited relatively broad range substrate specificity, oxidising a total of ten L-amino acids and, albeit to a much lesser extent, seven D-amino acids. Kinetic studies revealed that the three aromatic L-amino acids were the preferred substrates.