A new enzyme, N-alkylglycine oxidase, was isolated from a soil mold, Cladosporium sp. G-10. This protein, which was purified to near homogeneity by ammonium sulfate precipitation followed by successive column chromatography on phenyl-Sepharose, DEAE-Sepharose and Sephadex G-200, was a single polypeptide with a molecular mass of 52 000. In the presence of O 2 and H 2 O, this enzyme acted on some N-alkylglycine derivatives, such as N -carboxymethyllysine, N-carboxymethyl-6-aminocaproic acid, sarcosine and N-ethylglycine, and produced corresponding N-alkylamine, glyoxylic acid and H 2 O 2 . This enzyme had optimum activity at 30 o C, pH 8-10, and was most inhibited by ZnSO 4 , pCMB, iodoacetic acid, and SDS.