Substrate specificities of glycoside hydrolase families 8 (Rex), 39 (BhXyl39), and 52 (BhXyl52) beta-xylosidases from Bacillus halodurans C-125 were investigated. BhXyl39 hydrolyzed xylotriose most efficiently among the linear xylooligosaccharides. The activity decreased in the order of xylohexaose > xylopentaose > xylotetraose and it had little effect on xylobiose. In contrast, BhXyl52 hydrolyzed xylobiose and xylotriose most efficiently, and its activity decreased when the main chain became longer as follows: xylotetraose > xylopentaose > xylohexaose. Rex produced O-beta-D-xylopyranosyl-(1 -> 4)-[O-alpha-L-arabinofuranosyl-(1 -> 3)]-O-beta-D-xylopyranosyl-(1 -> 4)-beta-D-xylopyranose (Ara(2)Xyl(3)) and O-beta-D-xylopyranosyl-(1 -> 4)-[O-4-O-methyl-alpha-D-glucuronopyranosyl-(l -> 2)]-beta-D-xylopyranosyl-(1 -> 4)-beta-D-xylopyranose (MeGlcA(2)Xyl(3)), which lost a xylose residue from the reducing end of O-beta-D-xylopyranosyl-(1 -> 4)-[O-alpha-L-arabinofuranosyl-(1 -> 3)]-O-beta-D-xylopyranosyl-(1 -> 4)-beta-D-xylopyranosyl-(1 -> 4)-beta-D-xylopyranose (Ara(3)Xyl(4)) and O-beta-D-xylopyranosyl-(1 -> 4)-[O-4-O-methyl-alpha-D-glucuronopyranosyl-(1 -> 2)]-beta-D-xylopyranosyl-(1 -> 4)-beta-D-xylopyranosyl-(1 -> 4)-beta-D-xylopyranose (MeGlcA(3)Xyl(4)). It was considered that there is no space to accommodate side chains at subsite -1. BhXyl39 rapidly hydrolyzes the non-reducing-end xylose linkages of MeGlcA(3)Xyl(4), while the arabinose branch does not significantly affect the enzyme activity because it degrades Ara(3)Xyl(4) as rapidly as unmodified xylotetraose. The model structure suggested that BhXyl39 enhanced the activity for MeGlcA(3)Xyl(4) by forming a hydrogen bond between glucuronic acid and Lys265. BhXyl52 did not hydrolyze Ara(3)Xyl(4) and MeGlcA(3)Xyl(4) because it has a narrow substrate binding pocket and 2- and 3-hydroxyl groups of xylose at subsite +1 hydrogen bond to the enzyme.