A new flavoenzyme using molecular oxygen to oxidize L-glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2-oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including D-glutamic acid, L-glutamine and L-aspartic acid, only L-glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273,355 and 457 nm and the isoelectric point is at pH 6.2.The K,,, value for L-glutamic acid in air-saturated phosphate pH 7.0 was estimated to be 1.1 mM, the K,,, for oxygen was calculated to be 1.86 mM at saturating concentration of L-glutamic acid. The enzymic reaction is inhibited by Ag' and Hg2+ ions. The enzyme described here distinctly differs from two microbial L-glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure. . Even if the biological importance of the more specific L-amino acid oxidases is still unknown, they can serve as valuable analytical tools for the determination of L-amino acids. In particular, the specific determination of Lglutanlate would provide new possibilities for the estimation of serum glutamate oxaloacetate transaminase and glutamate pyruvate transaminase in the clinical laboratory and is also of interest for evaluating the quality of foods.The hitherto described L-glutamate oxidases from Streptomycetes catalyze the oxidation of L-glutamic acid to 2-oxoglutaric acid, simultaneously generating NH3 and H 2 0 2 . However, the enzyme from S. violuscens oxidizes Lglutamine and L-histidine [13] in addition to L-glutamate and the enzyme from S. X-119-6 oxidizes L-aspartic acid [14] to some extent.In this paper we report on the isolation, purification and characterization of a novel L-glutamate oxidase from the culture filtrate of Streptomyces endus, which is entirely specific for the conversion of L-glutamic acid.