D-amino acid oxidase was purified 285-fold from the yeast Rhodosporidium toruloides with a yield of 21%. The native molecular mass of the D-amino acid oxidase was 72 kDa with a subunit molecular weight of 37 kDa. It is a flavoprotein with one flavin adenine dinucleotide in each subunit as the prosthetic group. The optimal temperature was 35 degrees C and the optimal pH was 9.0. The enzyme was specific for D-amino acids. It was inhibited by 72, 49, and 21% in the presence of p-aminobenzoic acid, benzoic acid, and nicotinic acid, respectively. It showed high activity against cephalosporin C with a K-m of 0.65 mM.