1. The reaction of glucose oxidase [EC 1.1.3.4, β-D-glucose: oxygen oxidoreductase] obtained from Aspergillus niger was investigated by overall reaction kinetics as well as by the “stopped flow” and “rapid flow” methods. The experiments were carried out using D-glucose and 2-deoxy-D-glucose as substrates at pH 5.5 and at different temperatures (15°, 20°, 25°, and 30°C). 2. The difference absorption spectrum of the enzyme (steady state minus reduced level), which was obtained by the “stopped flow” method, was the same as that of the oxidized minus reduced level. No ESR-signal was observed within 5 msec, after mixing the oxidized enzyme solution with D-glucose or the reduced form with molecular oxygen. 3. When D-glucose or 2-deoxy-D-glucose was used as substrate, the value of kmaxred, the rate constant for reduction of the FAD moiety of oxidized enzyme at a sufficient concentration of the substrate, was almost the same as that of respective Vm/e obtained by overall reaction kinetics, (Vm/e: the maximum velocity per unit enzyme concentration). The value of Km at 25°C for 2-deoxy-D-glucose was the same as that for D-glucose at the same temperature. 4. The following scheme for the action mechanism of the glucose oxidase of Aspergillus niger was proposed to account for the data obtained. Eox+S ⇀Eox S ⇀ Ered+ P Ered+O2 ⇀ Eox+ H2O2 Where Eox stands for the oxidized form of the enzyme, EoxS the enzyme (oxidized form)-substrate compound, Ered the reduced form of the enzyme, S the substrate (D-glucose or 2-deoxy-D-glucose) and P the product (δ-lactone).