This work describes the purification and characterization of enzymes that exhibit b-D-xylosidase activity in stem tissues of Arabidopsis. This is the first detailed investigation that concerns the characterization of catalytic properties and sequence identity of enzymes with b-D-xylosidase activities in a dicotyledonous plant. Three different enzymes, ARAf, XYL4, and XYL1 with apparent molecular masses of 75, 67, and 64 kD, respectively, were purified to homogeneity. ARAf was identified as a putative a-L-arabinofuranosidase, and XYL4 and XYL1 as putative b-D-xylosidases using matrix-assisted laser-desorption ionization time of flight. ARAf belongs to family 51 and XYL4 and XYL1 to family 3 of glycoside hydrolases. ARAf and XYL1 have highest specificity for p-nitrophenyl-a-L-arabinofuranoside and XYL4 for p-nitrophenyl-b-D-xylopyranoside and natural substrates such as xylobiose and xylotetraose. XYL4 was shown to release mainly D-Xyl from oat spelt xylan, rye arabinoxylan, wheat arabinoxylan, and oligoarabinoxylans. ARAf and XYL1 can also release D-Xyl from these substrates but less efficiently than XYL4. Moreover, they can also release L-Ara from arabinoxylans and arabinan. Overall, the results indicate that XYL4 possesses enzymatic specificity characteristic for a b-D-xylosidase, while ARAf and XYL1 act as bifunctional a-L-arabinofuranosidase/b-D-xylosidases. Analysis of the activity of these three enzymes in stem tissues at different stages of development has shown that young stems possess the highest activities for all three enzymes in comparison to the activities of the enzymes present in stems at older stages of development. High enzyme activities are most likely related to the necessary modifications of cell wall structure occurring during plant growth. Plant cells are surrounded by an extracellular matrix known as the cell wall. The regulation of cell wall morphology and composition is important for the determination of cell size and shape, cellular interactions with the environment, mechanical resistance, and defense against pathogen attacks (Reiter, 2002). The cell wall is also involved in plant growth and development (Stolle-Smits et al., 1999;Obel et al., 2002). In addition, although cell walls consist mainly of polysaccharides broadly classified as celluloses, hemicelluloses, and pectins, their constitution varies, not only from plant to plant, but also in different tissues of the same plant (Heredia et al., 1995;Cosgrove, 1997;Popper and Fry, 2003). A coordinated series of biochemical processes occur during plant development resulting in the biosynthesis and degradation of cell wall components. Consequently, numerous enzymes must be implicated in these processes (Heredia et al., 1995;Cosgrove, 1997;Stolle-Smits et al., 1999;Obel et al., 2002;Reiter, 2002;Popper and Fry, 2003). However, to date, little information is available concerning such enzymes, their mode of action, and their physiological role. Increased research interest has focused on enzymes involved in the metabolism of hemicelluloses in ...