Tyrosine sulfate residues were identified in fibromodulin produced by tracheal chondrocytes, by tendon and sclera fibroblasts in primary culture, as well as in Chinese hamster ovary cells transfected with a construct containing fibromodulin cDNA. The tyrosine sulfate residues were located in the N-terminal part of fibromodulin. Thus, Chinese hamster ovary cells expressing a deleted variant of fibromodulin lacking the N-terminal 52 amino acids following the predicted signal peptide did not contain any tyrosine sulfate residues. The substitution with keratan sulfate chains was not restricted to chondrocytes, but was also identified in fibromodulin synthesized by bovine tendon fibroblasts and sclera fibroblasts, as well as in fibromodulin isolated from tendon. Digestion of fibromodulin with N-glycosidase F reduced the apparent size of fibromodulin to that of the core protein, as predicted from sequence analysis (Oldberg, A., Antonsson, P., Lindblom, K., and Heinegård, D. (1989) EMBO J.8, 2601-2604). Thus fibromodulin from cartilage, tendon, and sclera contains N-glycosidically linked oligosaccharides, some of which are extended to keratan sulfate chains.