Catalytic amino acid residues in a 1,3-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) and a homologous 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from barley have been investigated. To identify amino acids responsible for protonation of the glycosidic oxygen during hydrolysis, carbodiimide-mediated labeling of the enzymes with [14C]glycine ethyl ester was performed. This resulted in loss of activity and specific modification of the Glu288 residues in both enzymes. The stoichiometry of labeling was approximately 1:1, and modification was reduced in the presence of substrate analogues. Based on these data, the Glu288 residues are likely to be present at the active sites of the respective enzymes and may represent the catalytic acids in the hydrolytic reaction. The catalytic nucleophiles of the two enzymes were investigated by labeling with specific, mechanism-based epoxyalkyl-beta-oligoglucosides. Amino acid residues Glu232 and Glu231 were identified as the likely catalytic nucleophiles in the 1,3-1,4- and 1,3-beta-glucanases, respectively. Thus the position of the catalytic nucleophile and the putative proton donating amino acids in the two classes of beta-glucan endohydrolases are conserved. The acquisition of distinct substrate specificities in the evolution of these related enzymes may therefore not require the recruitment of novel catalytic amino acids but rather differences in their positioning at the active site and/or changes in substrate binding residues.