Peptide O-xylosyltransferase (EC 2.4.2.26) is the first enzyme required for the generation of chondroitin and heparan sulfate glycosaminoglycan chains of proteoglycans. Cloning of cDNAs has previously shown that, whereas invertebrates generally have a single xylosyltransferase gene, vertebrate genomes encode two similar proteins, xylosyltransferase I and II (XT-I and XT-II). To date, enzymatic activity has only been demonstrated for the human XT-I, Caenorhabditis SQV-6, and Drosophila OXT isoforms. In the present study, we demonstrate that a soluble form of human XT-II expressed in the xylosyltransferase-deficient pgsA-745 (S745) Chinese hamster ovary cell line is indeed capable of catalyzing the transfer of xylose to a variety of peptide substrates; its enzyme activity was also proven using a Pichia-expressed form of XT-II. Its pH, temperature, and cation dependences are similar to those of XT-I expressed in either mammalian cells or yeast. Our data suggest that XT-I and XT-II are, at least in vitro, functionally identical.