We have reported that 3-estradiol-beta-D-xyloside primes heparan sulfate synthesis in Chinese hamster ovary cells and that the proportion of heparan sulfate made rises with increasing concentration of xyloside (Lugemwa, F.N. and Esko, J.D. (1991) J. Biol. Chem. 266, 6674-6677). Using estradiol as a guide, we varied the structure of the aglycone and showed that beta-D-xylosides containing two fused aromatic rings efficiently prime heparan sulfate. Thus, 2-naphthol-beta-D-xyloside primed heparan sulfate at low dose (< or = 10 microM) and the proportion of heparan sulfate increased with concentration (up to 50% of total glycosaminoglycan). Various ring additions and heterocyclic ring substitutions altered the efficiency of heparan sulfate priming, but had no effect on the overall level of glycosaminoglycan synthesis. Replacement of the bridging oxygen with sulfur (2-naphthalenethiol-beta-D-xyloside) increased the efficiency of heparan sulfate priming. Priming of heparan sulfate correlated with hydrophobicity of the xyloside, but several exceptions suggested that the chemical structure of the aglycone played an equally important role. Interestingly, the heparan sulfate chains generated on 2-naphthol-beta-D-xyloside showed a 2-fold decrease in the proportion of disaccharides containing 6-O-sulfate groups and a striking diminution in non-sulfated iduronic acid containing disaccharides compared to the chains attached to cellular proteoglycans. Thus, both the type of glycosaminoglycan made on a xyloside and its fine structure depends on the aglycone.