Xylooligosaccharide (XO) production was performed from xylan, which was obtained by alkali extraction from cotton stalk, a major agricultural waste in Turkey. Enzymatic hydrolysis was selected to prevent byproduct formation such as xylose and furfural. Xylan was hydrolyzed using a commercial xylanase preparation, and the effects of pH, temperature, hydrolysis period, and substrate and enzyme concentrations on the XO yield and degree of polymerization (DP) were investigated. Cotton stalk contains about 21% xylan, the composition of which was determined as 84% xylose, 7% glucose, and 9% uronic acid after complete acid hydrolysis. XOs in the DP range of 2-7 (X6 approximately X5>X2>X3) were obtained with minor quantities of xylose in all of the hydrolysis conditions used. Although after 24 h of hydrolysis at 40 degrees C, the yield was about 53%, the XO production rate leveled off after 8-24 h of hydrolysis. XO yield was affected by all of the parameters investigated; however, none of them affected the DP of the end product significantly, except the hydrolysis period. Enzyme hydrolysis was maintained by the addition of fresh substrate after 72 h of hydrolysis, indicating the persistence of enzyme activity. The optimal hydrolysis conditions were determined as 40 degrees C, pH 5.4, and 2% xylan. The obtained product was fractionated via ultrafiltration by using 10, 3, and 1 kDa membranes. Complete removal of xylanase and unhydrolyzed xylan was achieved without losing any oligosaccharides having DP 5 or smaller by 10 kDa membrane. After a two-step membrane processing, a permeate containing mostly oligosaccharides was obtained.