A specific and sensitive substrate for the assay of endo-1,4-beta-glucanase (cellulase) has been prepared. The substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-beta-cellotrioside (BzCNPG3) in the presence of thermostable beta-glucosidase. Hydrolysis by exo-acting enzymes such as beta-glucosidase and exo-beta-glucanase is prevented by the presence of the benzylidene group on the non-reducing end D-glucosyl residue. On hydrolysis by cellulase, the 2-chloro-4-nitrophenyl-beta-glycoside is immediately hydrolysed to 2-chloro-4-nitrophenol and free D-glucose by the b-glucosidase in the substrate mixture. The reaction is terminated and colour developed by the addition of a weak alkaline solution. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation. This procedure should find widespread applications in biomass enzymology and in the specific assay of endo-1,4-beta-glucanase in general. (C) 2013 Elsevier Ltd. All rights reserved.