A method for isolation of D-amino acid oxidase (DAAO) from disrupted Trigonopsis variabilis cells has been developed. In an aqueous two-phase system consisting of PEG6000 (220 g l(-1)), potassium phosphate (110 g l(-1), K2HPO4 + KH2PO4 = 10.1:1, mol mol(-1)) and dl-methionine (11 g l(-1)), the major portion of cellular proteins (87%) was partitioned into the salt phase. By sequential extraction, 48% of DAAO was recovered in PEG phase, giving a yield of 211 U mg protein(-1).