The production level of pyranose oxidase (pyranose : oxygen oxidoreductase, EC 1.1.3.10), which is recently drawing increasing attention as an enzyme for the specific diagnostic analysis of diabetes mellitus, was found to be markedly elevated by investigating the cultural conditions of its producer, basidiomycetous fungus no. 52. When the strain was cultivated at 20 degrees C for 142 h in medium consisting of 1% lactose, 4% soybean meal, 1% cornsteep liquor and 0.1% L-leucine in tap water (pH 6.0) with feeding of 1% lactose three times during the course of cultivation in a 5-l jar fermenter, the total and specific activities of intracellular pyranose oxidase reached maximum values (13 units/ml of culture broth and 1.9 units/mg protein, respectively). These values were 45- and I-fold higher, respectively, than those obtained previously (Ref. 9), and are considered to be satisfactory for industrial production.