BACKGROUND: High-temperature enzymatic hydrolysis of cellulose has been studied recently with promising results. High temperatures can improve reaction rates and mass transfer, and lower viscosity. Tests were made to determine how short high-temperature prehydrolysis steps affect the final hydrolysis with commercial cellulase mixture. RESULTS: Continuous mixing improved the hydrolysis of 3% Avicel by Pyrococcus horikoshii endoglucanase (PhEG) at 70 degrees C but not at 100 degrees C. Differences in the temperature effect between 90 degrees C and similar to 103 degrees C were much higher than between 70 degrees C and 90 degrees C. It was essential that the dosed enzyme activity was the same at each temperature, meaning lower volumetric amount of enzyme at higher temperature. PhEG prehydrolysis (2 h) of steam pretreated spruce before final hydrolysis by commercial cellulase mixture was more efficient at 100 degrees C than at 80 degrees C, improving the total enzymatic hydrolysis of cellulose by 15-30%. Xylanase and mannase prehydrolysis at 80 degrees C (2 h) improved the cellulase hydrolysis by 20-33%, while all three enzymes together improved the final enzymatic yield by 50-70%. CONCLUSION: A temperature of 100-105 degrees C provides clear process advantages for enzymatic treatments when compared with 70-90 degrees C. Enzymatic high-temperature prehydrolysis could be applicable to circumvent the problems of high viscosity and to reduce the required amount of cellulases in the subsequent hydrolysis. (C) 2015 Society of Chemical Industry