Chondroitin and heparan sulfates are essential players in animal development and are synthesized by a series of glycosyltransferases, the first of which is UDP-alpha-D-xylose:proteoglycan core protein beta-D-xylosyltransferase (EC ). In the present study, a Drosophila melanogaster gene (CG17771), previously designated as a homologue of core 2 and I beta1,6-N-acetylglucosaminyltransferases, was shown to encode an active peptide O-xylosyltransferase. A novel coupled assay using matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated transfer of xylose to the peptide DDDSIEGSGGR. Analysis of sequences of various peptide O-xylosyltransferase and beta1,6-N-acetylglucosaminyltransferase sequences indicates that they are members of a large multifunctional protein family with a range of roles in beta-glycosylation of either peptide or glycan substrates. Because in contrast to mammals, there is only one fly peptide O-xylosyltransferase gene, it is anticipated that, given the key roles of proteoglycans, the hereby designated oxt gene is essential for viability.