The maintenance of human osteoarthritic femoral head cartilage in long-term explant culture has been used to assess changes in newly synthesized and endogenous proteoglycans. Minced cartilage was radiolabeled with [3H]-leucine for 24 h. and the high density proteoglycans digested with tosyl-phenylalanine chloromethyl ketone-trypsin. Chondroitin-sulfate-rich peptides were separated from chondroitin-sulfate-poor peptides by DEAE-cellulose chromatography and hexuronic acid, protein, and galactosamine and glucosamine molar ratios determined. The incorporation of [3H]-leucine was highest in peptides enriched in chondroitin sulfate. when day 1 cultures were compared to those maintained for 20 days, several prominent changes were seen including, a reduction in the galactosamine to glucosamine ratio. Taken together with other data which showed a reduction in the hydrodynamic size of these proteoglycans with time in culture, these results showed that changes in the existing extracellular matrix pertinent to the osteoarthritic process can be assessed by maintaining cartilage in long-term organ-explant culture. A reduced hydrodynamic size of newly synthesized proteoglycans is consistent with a loss of chondroitin-sulfate and an increase in keratan sulfate in the high density proteoglycans.