A two-stage biomass pretreatment process-a combination of autohydrolysis and aqueous ammonia percolation-was experimentally studied as a method to remove and recover hemicellulose from lignocellulosic biomass. Hemicellulose was completely separated from the biomass after 1 hr of autohydrolysis at 200℃. As reaction temperature and/or time of autohydrolysis was increased in the range of 170-200℃ and 1-2.5 hr, respectively, the amount of hemicellulose solubilization was increased; however, more sugars were decomposed. Most of the extracted hemicellulose was recovered as xylose oligomer. Hemicellulose was found to inhibit the enzymatic hydrolysis of cellulose. When the biomass was consecutively pretreated with pure water at 180℃ for 30 min and with 10 wt% ammonia solution at 180℃ for 30 min, about 62% of the hemicellulose was extracted. The enzymatic digestibility of the pretreated biomass was as high as 95%.