The general properties and specificity of a dextran alpha-(1 yields 2)-debranching enzyme from Flavobacterium have been examined in order to apply this enzyme to the structural analysis of highly branched dextrans. The optimum pH range and temperature were pH 5.5-6.5, and 45 degrees, respectively. The enzyme was stable up to 40 degrees on heating for 10 min, and over a pH range of 6.5-9.0 on incubation at 4 degrees for 24 h. The effects of various metal ions and chemical reagents have also been examined. The debranching enzyme has a strict specificity for the (1 yields 2)-alpha-D-glucosidic linkage at branch points of dextrans and related branched oligosaccharides, and produces D-glucose as the only reducing sugar. The degree of hydrolysis of the dextrans by this enzyme and the Km value (mg/mL) were as follows: B-1298 soluble, 25.2%, 0.21; B-1299 soluble, 31.5%, 0.27; and B-1397, 11.8%, 0.91. the debranching enzyme thus has a novel type of specificity as a dextranhydrolase. We have termed this enzyme as dextran alpha-(1 yields 2)-debranching enzyme, and its systematic name is also discussed.