The specificity of the glycosaminoglycan (GAG) acceptor sites of ryudocan was examined by stably expressing epitope-tagged ryudocan cDNA constructs in mouse L cells, which normally produce this proteoglycan. Immunopurified ryudocan was glycanated with both heparan sulfate (HS) and chondroitin sulfate (CS). The attachment of GAGs to ryudocan was prevented by creating Ser-->Thr mutations in all possible combinations at positions 44, 65, and 67. The resulting ryudocan of exogenous origin was immunopurified and evaluated with regard to attached GAG chains. The data reveal that ryudocan possesses three functional GAG attachment sites, that the sites are always occupied with GAG chains, and that each site is capable of bearing either HS or CS. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of GAG lyase digests of intact ryudocan reveal the production of the following multiple isoforms: pure HS-ryudocan, various HS/CS-hybrids, and pure CS-ryudocan. The data suggest that the occupancy bias of each site for HS or CS is slight and that each site functions in a relatively independent fashion. The GAG lyase analysis of partially purified L cell proteoglycans shows two pure CS-homoglycans with core proteins of M(r) = 130,000 and 52,000, respectively. A similar analysis of immunopurified L cell glypican demonstrates that this species only exists as a pure HS-homoglycan. The production of pure homoglycans by this clonal cell line strongly suggests that the functional promiscuity of GAG attachment sites of ryudocan must be encoded in the core protein structure. This property of ryudocan is not peculiar to L cells, as ryudocan synthesized by early passage human endothelial cells also bears both HS and CS. The production of multiple isoforms of ryudocan may serve to expand the functional versatility of this cell surface component and allow it to participate in many different biologic processes.