D-Amino acid oxidase activities for D-glutamate (D-Glu), D-aspartate (D-Asp) and D-alanine (D-Ala) were found in cell-free extract of Aspergillus sojae (A. sojae). The enzyme activities for these three substrates increased over 30-fold by the addition of 0.25% D-Ala to the culture medium. Glycerol was an effective carbon source for increasing the enzyme activities. D-Ala, D-serine (D-Ser), and D-tryptophan (D-Trp) were better inducers than other D-amino acids. D-Glu and D-Asp were oxidized at rates of 70 and 6%, respectively, relative to the rate of oxidation of D-Ala which was taken as 100%. A. sojae D-amino acid oxidase showed no inhibition by sodium benzoate or dicarboxylates and had a molecular weight of 129,000, which differed substantially from those of D-amino acid oxidases of porcine and rabbit kidney.