Endoglucanases are important enzymes for biomass conversion and other industrial processes. Determining the specificity of endoglucanases from various glycoside hydrolase families is of interest for bioinformatic functional prediction and substrate-tailored enzyme development. To do so, we characterized similar to 30 endoglucanases from six glycoside hydrolase families. For p-nitrophenyl cellobioside and lactoside, only family 7 enzymes showed significant activity. For xyloglucan, both family 7 and 12 enzymes showed significant activity. For xylan and arabinoxylan, only family 7 enzymes showed significant activity. For mannan and galactomannan, both family 5 and 9 enzymes showed significant activity. The difference in specificity was preliminarily attributed mainly to the structural difference of the enzymes' active sites. For family 7 endoglucanases, difference in thermal stability might affect their performance in hydrolyzing various (hemi)cellulose substrates. Phylogenetic analysis on the subfamily distribution of family 5 endoglucanases (in relation with mannanases) suggested that their mannanase side-activity might be the remnant of an ancestral multi-function enzyme. Similar analysis was also made with the xyloglucanase or arabionxylans side-activity of family 12 and 7 endoglucanases. The apparent dependence of the specificity on family (primary/tertiary structure) might assist us in better understanding the structure-function relationship of the enzymes, and developing more versatile biocatalysts for the utilization of biomass. (C) 2009 Elsevier Ltd. All rights reserved.