Synergism between cellulases facilitates efficient hydrolysis of microcrystalline cellulose. We hypothesize that the effects of synergism, observed as enhanced extents of hydrolysis, are related to cellulase binding to the substrate in mixtures. In this study, direct measurements of bound concentrations of fluorescence-labeled T fusca Cel5A, Cel6B, and Cel9A on bacterial microcrystalline cellulose were used to study binding behaviors of cellulases in binary component reactions. The accuracy of the determination of fluorescence-labeled cellulase concentrations in binary component mixtures was in the range of 7-9%. Data at 5 degreesC show that binding levels of cellulases in mixture reactions are only 22-70% of the binding levels in single component reactions. At 50 degreesC, however, most of the cellulase components in the same mixtures bound to extents of 40-126% higher than in the corresponding single component reactions. The degrees of synergistic effect (DSE) observed for the reactions at 50 degreesC were greater than 1, indicating that the components in the mixture acted synergistically, whereas DSE < 1 was generally observed for the reactions at 5 degreesC indicating anti-synergistic behavior. Degrees of synergistic binding (DSB) were also calculated, where anti-synergistic mixtures had DSB < 1 and synergistic mixtures had DSB > 1. We conclude that the lower extents of binding at 5 degreesC are due to competition for binding sites by the cellulase components in the mixtures and the enhanced binding extents at 50 degreesC are due to increased availability of binding sites on the substrates brought about by the higher extents of hydrolysis.