Steam refining was investigated as a pretreatment for enzymatic hydrolysis of poplar wood from a short rotation plantation. The experiments were carried out without debarking to use an economically realistic raw material. Steam refining conditions were varied in the range of 3-30 min and 170-220 degrees C, according to a factorial design created with the software JMP from SAS Institute Inc., Cary, NC, USA. Predicted steaming conditions for highest glucose and xylose yields after enzymatic hydrolysis were at 210 degrees C and 15 min. Control tests under the optimized conditions verified the predicted results. Further pretreatments without bark showed that the enzymes were not significantly inhibited by the bark. The yield of glucose and xylose was 61.9% of theoretical for the experiments with the whole raw material, whereas the yield for the experiments without bark was 63.6%. Alkaline extraction of lignin from the fibers before enzymatic hydrolysis resulted in an increase of glucose yields from mild pretreated fibers and a decrease for severe pretreated fibers. The extracted lignin had a high content of xylose of up to 14% after very mild pretreatments. On the other hand, molecular weights of the extracted lignin increased substantially after pretreatments with a severity factor above 4. Hence, alkaline extraction of the lignin seems only attractive in a narrow range of steaming conditions.