Lignin-derived inhibition is a major obstacle restricting the enzymatic hydrolysis of cell wall polysaccharides especially with softwood lignocellulosics. Enzyme adsorption on lignin is suggested to contribute to the inhibitory effect of lignin. The interaction of cellulases with softwood lignin was studied in the present work with commercial Trichoderma reesei cellulases (Celluclast) and lignin-rich residues isolated from steam pretreated softwood (SPS) by enzymatic and acid hydrolysis. Both lignin preparations inhibited the hydrolysis of microcrystalline cellulose (Avicel) and adsorbed the major cellulases present in the commercial cellulase mixture. The adsorption phenomenon was studied at low temperature (4°C) and at the typical hydrolysis temperature (45°C) by following activities of free and lignin-bound enzymes. Severe inactivation of the lignin-bound enzymes was observed at 45°C, however at 4°C the enzymes retained well their activity. Furthermore, SDS–PAGE analysis of the lignin-bound enzymes indicated that very strong interactions form between the residue and the enzymes at 45°C, because the enzymes were not released from the residue in the electrophoresis. These results suggest that heat-induced denaturation may take place on the surface of softwood lignin at the hydrolysis temperature. Biotechnol. Bioeng. 2011;108: 2823–2834. © 2011 Wiley Periodicals, Inc.