Xyloglucan is the main hemicellulose in the primary cell walls of most seed plants and is thought to play a role in regulating the separation of cellulose microfibrils during growth. Xylose side chains block the degradation of the backbone, and a-xylosidase activity is necessary to remove them. Two Arabidopsis (Arabidopsis thaliana) mutant lines with insertions in the a-xylosidase gene AtXYL1 were characterized in this work. Both lines showed a reduction to undetectable levels of a-xylosidase activity against xyloglucan oligosaccharides. This reduction resulted in the accumulation of XXXG and XXLG in the liquid growth medium of Atxyl1 seedlings. The presence of XXLG suggests that it is a poor substrate for xyloglucan b-galactosidase. In addition, the polymeric xyloglucan of Atxyl1 lines was found to be enriched in XXLG subunits, with a concomitant decrease in XXFG and XLFG. This change can be explained by extensive exoglycosidase activity at the nonreducing ends of xyloglucan chains. These enzymes could thus have a larger role than previously thought in the metabolism of xyloglucan. Finally, Atxyl1 lines showed a reduced ability to control the anisotropic growth pattern of different organs, pointing to the importance of xyloglucan in this process. The promoter of AtXYL1 was shown to direct expression to many different organs and cell types undergoing cell wall modifications, including trichomes, vasculature, stomata, and elongating anther filaments.