Xyloglucan (XyG) is the principal hemicellulose found in the primary cell walls of most plants. XyG is composed of a beta-(1,4)-glucan backbone that is substituted in a regular pattern with xylosyl residues, which are added by at least one and likely two or three xylosyltransferase (XT) enzymes. Previous work identified seven Arabidopsis thaliana candidate genes, one of which (AtXT1) was shown to encode a protein with XT activity (Faik, A., Price, N. J., Raikhel, N. V., and Keegstra, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7797-7802). We expressed both AtXT1 and a second closely related gene, now called AtXT2, in insect cells and demonstrated that both have XT activity for cellopentaose and cellohexaose acceptor substrates. Moreover, we showed that cellohexaose was a significantly better acceptor substrate than cellopentaose. Product structural characterization showed that AtXT1 and AtXT2 preferentially added the first xylosyl residue to the fourth glucosyl residue from the reducing end of both acceptors. Furthermore, when the ratio of UDP-xylose to cellohexaose and the reaction time were increased, both AtXT1 and AtXT2 added a second xylosyl residue adjacent to the first, which generated dixylosylated cellohexaose. On the basis of these results, we concluded that AtXT1 and AtXT2 have the same acceptor specificities and generate the same products in vitro. The implications of these results for understanding in vivo XyG biosynthesis are considered.