The formation of chondroitin sulfate is initiated by xylosyltransferase (XT) transferring xylose from UDP-xylose to consensus serine residues of proteoglycan core proteins. Our alignment of 51 amino acid sequences of chondroitin sulfate attachment sites in 19 different proteins resulted in a consensus sequence for the recognition signal of XT. The complete recognition sequence is composed of the amino acids a-a-a-a-G-S-G-a-b-a, with a = E or D and b = G, E, or D. This sequence was confirmed by determination of the Michaelis-Menten constants for in vitro xylosylation of different synthetic proteins and peptides using an enriched XT preparation from conditioned cell culture supernatant of human chondrocytes. The highest acceptor activity was determined by the sequence Q-E-E-E-E-G-S-G-G-G-Q, which was found in the single chondroitin sulfate attachment site of bikunin, the inhibitory active component of the human inter-alpha-trypsin inhibitor. We determined the Michaelis-Menten constant (Km) of xylosylation of the synthetic bikunin analogous peptide Q-E-E-E-G-S-G-G-G-Q-K to be 22 microM, which was 9-fold decreased in comparison to deglycosylated core protein from bovine cartilage (188 microM), which was previously used as acceptor for the XT activity assay. The best XT acceptors were nonglycosylated recombinant wild-type bikunin (Km = 0. 9 microM) and the recombinant [Val36,Val38]delta1,[Gly92, Ile94]delta2bikunin (Km = 0.6 microM), a variant without any inhibitory activity against serine proteinases. These results imply that the primary structure of the acceptor is not the only determinant for recognition by xylosyltransferase. Thus, protein conformation is also a main factor in determining xylosylation.