In order to study proteoglycan retention and accumulation, we optimized a chondrocyte cell culture system in which isolated bovine articular chondrocytes accumulate extracellular matrix to form a continuous layer of cartilagenous tissue. The tissue can attain a thickness of up to 110 microns by 35 days. The cells synthesize large keratan sulfate containing proteoglycans and type II collagen indicating that the chondrocytes maintain their phenotype in these culture conditions. Matrix accumulation is enhanced by increased cell density and the presence of serum and ascorbic acid. The amount of proteoglycans synthesized by the chondrocytes increases up to day 21 and then decreases to the same levels as are synthesized during the first week of culture. The percentage of newly synthesized proteoglycans retained in the matrix increases from 20% on day 6 to a maximum of 85% by day 35. The proteoglycan and collagen content in the tissue increases with time in culture. The changes in the percentage of proteoglycans retained parallels the increase in proteoglycan content. After day 35, there is no further increase in the amount of proteoglycans and collagen nor in the percentage of newly synthesized proteoglycans retained in the extracellular matrix. These studies demonstrate that the cultures are going through two phases: one of matrix accumulation and then one of maintaining the existing matrix. The period of matrix accumulation occurs between days 10-21 whereas matrix maintenance is observed after day 35. Using this culture system to study proteoglycan accumulation and maintenance during these culture periods may prove useful in identifying the mechanisms regulating these processes.