A two-step liquid hot water pretreatment (TSLHW) was developed with the objective of achieving complete saccharification of both hemicellulose and cellulose of Eucalyptus grandis, thereby avoiding the problems associated with the use of strong acid catalysts. The first step of the pretreatment was studied in the temperature range 180-200 degrees C, and the highest yield of total xylose achieved was 86.4% after 20 min at 180 degrees C. The second-step of the pretreatment was studied in the temperature range 180-240 degrees C and for lengths of time of 0-60 min. The conversion rate of glucan was more sensitive to temperature than time. The optimum reaction conditions for the second step of the pretreatment with minimal degradation of sugars were 200 degrees C for 20 min. the total sugar recovery from E. grandis with the optimized pretreatment and 72 h enzymatic digestion, reached 96.63%, which is superior to the recovery from a single-step pretreatment with hot water or dilute acid. (C) 2009 Elsevier Ltd. All rights reserved.