A glycerol oxidase from Aspergillus japonicus oxidized ethylene glycol to glyoxal by the same reaction pathway as alcohol oxidases from methanol yeast. The optimum pH and temperature for the oxidation of ethylene glycol were around 7.0 and 40 degrees C, respectively. Those of glycolaldehyde were similar to those of ethylene glycol. The apparent Kms for ethylene glycol and glycolaldehyde were 195 and 48.8 mM, respectively. The maximum velocities for ethylene glycol and glycolaldehyde were 89.1 and 62.2 mumol/min/mg of protein, respectively. Glycerol oxidase also oxidized glycolic acid, which is not oxidized by the alcohol oxidases, to glyoxylic acid like glycolate oxidases from green plants, and the apparent Km and Vmax for glycolic acid were 114 mM and 2.68 mumol/min/mg of protein, respectively. The glycerol oxidase was applicable to the production of glyoxal and glyoxylic acid.