The adsorption of cellulase enzymes onto lignin is shown to be non-productive and therefore reduces enzymatic hydrolysis of liquid hot water pretreated cellulose. Among the enzyme components of Trichoderma reesei cellulase cocktail, -glucosidase showed the strongest adsorption onto lignin. Only 2-18% of the initial -glucosidase activity remained in the supernatant while 50-60% of cellobiohydrolase and endoglucanase activities were recovered after incubation with lignin. By increasing the pH to 5.5 and adding NaCl to a 200mM, the free enzymes in the supernatant were increased but hydrolysis was not enhanced since optimal pH for enzymatic hydrolysis is at 4.8. Electrostatic interactions contributed to enzyme adsorption and their effect was most pronounced for T. reesei -glucosidase which had high molecular weights (78-94kDa) and high isoelectric points (pI 5.7-6.4). Since the enzyme components which are required to synergistically hydrolyze cellulose have different profiles (molecular weight, hydrophobicity and pI), they exhibit different adsorption behaviors with lignin, and thereby change the ratio of enzyme activities needed for synergism during cellulose hydrolysis. -glucosidase from Aspergillus niger exhibits less adsorption than -glucosidase from T. reesei. Supplemental addition of A. niger -glucosidase to the enzyme mixture increases hydrolysis of pretreated hardwood by a factor of two. The analysis presented in this paper shows that lignins with higher guaiacyl content adsorb more cellulase enzymes, particularly -glucosidase, and that adsorption of -glucosidase onto lignin indirectly suppresses enzymatic hydrolysis of cellulose in pretreated hardwoods due to decreased hydrolysis of cellobiose which in turn accumulates and inhibits CBH. Biotechnol. Bioeng. 2015;112: 447-456. (c) 2014 Wiley Periodicals, Inc.