An endoglucanase was isolated from cell walls of Zea mays seedlings. Characterization of the hydrolytic activity of this glucanase using model substrates indicated a high specificity for molecules containing intramolecular (1-->3),(1-->4)-beta-d-glucosyl sequences. Substrates with (1-->4)-beta-glucosyl linkages, such as carboxymethylcellulose and xyloglucan were, degraded to a limited extent by the enzyme, whereas (1-->3)-beta-glucans such as laminarin were not hydrolyzed. When (1-->3),(1-->4)-beta-d-glucan from Avena endosperm was used as a model substrate a rapid decrease in vicosity was observed concomitant with the formation of a glucosyl polymer (molecular weight of 1-1.5 x 10(4)). Activity against a water soluble (1-->3),(1-->4)-beta-d-glucan extracted from Zea seedling cell walls revealed the same depolymerization pattern. The size of the limit products would indicate that a unique recognition site exists at regular intervals within the (1-->3),(1-->4)-beta-d-glucan molecule. Unique oligosaccharides isolated from the Zea (1-->3),(1-->4)-beta-d-glucan that contained blocks of (1-->4) linkages and/or more than a single contiguous (1-->3) linkage were hydrolyzed by the endoglucanase. The unique regions of the (1-->3),(1-->4)-beta-d-glucan may be the recognition-hydrolytic site of the Zea endoglucanase.