CelZ from the cellulolytic thermophile Clostridium stercorarium has been described as a 'monomeric' cellulase able to effect both the endoglucanolytic hydrolysis of internal glycosidic linkages and the exoglucanolytic degradation from the chain ends in a processive mode of action. The putative catalytic residues of this family 9 cellulase, Asp84 and Glu447 located within the N-terminal domain of the modular protein, were replaced by site-directed mutagenesis. A minimized CelZ derivative (CelZC') comprising the catalytic domain and the adjacent cellulose-binding domain (CBD) family IIIc domain C' was used as target for mutagenesis. Six mutant enzymes and the unmodified CelZC' protein were purified to homogeneity and compared with respect to thermoactivity, substrate specificity, product profile and synergism. CD studies revealed that no major changes to the overall structure of the proteins had occurred. Replacement of either one or both catalytic residues completely eliminated the ability of CelZ to attack insoluble Avicel preparations indicative of the exo-activity, whereas the endo-activity measured via hydrolysis of CM-cellulose was retained upon substitution of the catalytic base Asp84. Thus, endo-active CelZ mutants defective in the exo-activity were available for co-operativity studies with the C. stercorarium exoglucanase CelY. Synergism was found to be dependent on the endo-activity of CelZ. Mutants Asp84Gly and Asp84Glu were able to enhance the degradation of crystalline cellulose significantly, although no products could be released from this substrate by individual action of the mutants.