A beta-d-glucanase highly specific for glucans containing a linkage sequence ... Glc 1 --> 4 Glc 1 --> 3 Glc 1 --> 4 Glc ... has been isolated from several commercial preparations of Bacillus subtilis alpha-amylase including one purified by repeated crystallization. The beta-d-glucanase will not hydrolyze cellulose or laminarin. Gel filtration on a Bio-Gel P-200 column results in separation of the glucanase from the alpha-amylase. The enzyme is of the endo type as changes in the substrate viscosity appear long before the appearance of detectable reducing sugars. No evidence of product inhibition was revealed and appropriate substrates were converted to oligosaccharides, the quantity of which approaches theoretical yields. The products of the reaction were separated according to molecular size by use of Bio-Gel P-2 gel filtration and found to be consistent with the action pattern of the enzyme. Kinetic studies show that the enzyme has an optimum activity at pH 6.5, a V(max) of 13.9 mug glucose equivalent released/mug protein.hour, and an apparent Km of 3.4 mg of lichenan per ml. Potential application of this enzyme for the structural characterization of plant cell wall glucans is discussed.