A membrane-bound glucoside 3-dehydrogenase [EC 1.1.99.13], which oxidizes validoxylamine A to the 3-keto derivative, was solubilized from the membrane fraction of Flavobacterium saccharophilum by Triton X-100 and purified about 280-fold with an overall yield of 30% from the membrane fraction by column chromatography on DEAE- and CM-Sepharose CL-6B and gel filtration on Sephacryl S-300. The purified enzyme exhibited a single protein band on disc gel electrophoresis, and FAD was shown to be the prosthetic group. The enzyme had a molecular weight of 270,000 as determined by gel filtration on Sephacryl S-300 and consisted of 4 identical subunits each with a molecular weight of 66,000. The enzyme reacted with various artificial electron acceptors such as 2,6-dichlorophenolindophenol (DCIP), phenazine methosulfate, and ferricyanide. The optimum pH for DCIP reductase activity was 6.0. The enzyme was inhibited by Hg2+ and p-chloromercuribenzoate. D-Glucose and methyl-alpha- and beta-D-glucoside showed the highest susceptibility to the enzyme, and were converted to the corresponding 3-keto sugars.