Alkaline pretreatment of spruce at low temperature in both presence and absence of urea was studied. It was found that the enzymatic hydrolysis rate and efficiency can be significantly improved by the pretreatment. At low temperature, the pretreatment chemicals, either NaOH alone or NaOH–urea mixture solution, can slightly remove lignin, hemicelluloses, and cellulose in the lignocellulosic materials, disrupt the connections between hemicelluloses, cellulose, and lignin, and alter the structure of treated biomass to make cellulose more accessible to hydrolysis enzymes. Moreover, the wood fiber bundles could be broken down to small and loose lignocellulosic particles by the chemical treatment. Therefore, the enzymatic hydrolysis efficiency of untreated mechanical fibers can also be remarkably enhanced by NaOH or NaOH/urea solution treatment. The results indicated that, for spruce, up to 70% glucose yield could be obtained for the cold temperature pretreatment (−15°C) using 7% NaOH/12% urea solution, but only 20% and 24% glucose yields were obtained at temperatures of 23°C and 60°C, respectively, when other conditions remained the same. The best condition for the chemical pretreatment regarding this study was 3% NaOH/12% urea, and −15°C. Over 60% glucose conversion was achieved upon this condition. Biotechnol. Bioeng. 2008;99: 1320–1328. © 2007 Wiley Periodicals, Inc.