Abstract The specificity of glucose oxidase for catalyzing the oxidation of β- d -glucose in contrast to α- d -glucose, other hexoses, and pentoses was investigated. The enzyme appears to catalyze the oxidation of the beta form only, and the mutarotation of the alpha form is rate limiting. d -Galactose, d -mannose, and d -xylose are slowly oxidized in the presence of glucose oxidase, but approximately 300 times as much enzyme is required to give a rate of oxidation equal to that for glucose. Galactose and mannose are oxidized directly to the corresponding aldonic acids without a prior conversion to glucose. Thirtyfold purification of the enzyme does not increase the ratio of glucose to galactose oxidation. d -Arabinose, which itself is not oxidized, inhibits glucose oxidase in catalyzing the oxidation of glucose. The clinical implications of these specificity studies are discussed.