An enzyme that oxidizes acidic D-amino acids was purified from the yeast Candida boidinii 2201 to homogeneity as indicated on SDS-polyacrylamide gel electrophoresis. The molecular mass determined by the electrophoresis was 45 kDa, while gel filtration of the native enzyme showed a molecular mass of 43 kDa, indicating the native enzyme to be a monomer. The enzyme required FAD for activity and FMN did not replace FAD. The purified preparation had a specific activity of 41.4 mu mol/min per mg protein with 20 mM D-glutamate as a substrate. The optimum pH and temperature were 7.0 and 37 degrees C. The K-m, for D-glutamate was higher than that for D-aspartate, while the k(cat) was more markedly higher for the former than for the latter, resulting in a higher k(cat)/K-m value for D-glutamate. This indicates that D-glutamate is the better substrate and suggests that the enzyme should be named D-glutamate oxidase (EC 1.4.3.7). N-Methyl-D-aspartate was a far poorer substrate than these two compounds. D-Malate was the most potent competitive inhibitor, followed by glutarate and meso-tartrate. The N-terminal sequence of the enzyme was similar to those of FAD enzymes, including D-aspartate and D-amino acid oxidases from various sources.