Human synovial cells in culture are known to synthesize hyaluronic acid, but the production of sulfated glycosaminoglycans (GAG) has received less attention. Using 14C-glucosamine as a precursor, GAG content was studied in the medium, trypsin-solubilized pericellular layer, and cell residue fraction of cultured synovial cells derived from the synovial membranes of nonrheumatoid and rheumatoid joints. Over 90% of the total non-dialyzable counts appeared in the culture medium, for the most part in hyaluronic acid. The remaining nondialyzable counts were cell-associated, almost equally divided between the pericellular layer and cell residues. In these fractions, only part of the counts were in GAG susceptible to testicular hyaluronidase digestion, and GAG were significantly lower in the cell residue of the rheumatoid synovial cells compared to the nonrheumatoid cells. Analysis of the chondroitinase ABC and AC digestion products of these GAG indicated the presence of chondroitin-4 and -6 sulfates, and dermatan sulfate, but not heparan sulfate. Similar findings with respect to the identity of the GAG in nonrheumatoid and rheumatoid synovial cell culture media were obtained with 35SO4 as a precursor.