Proteoglycans synthesized by chondrocytes in alginate beads are found in two compartments: the cell-associated matrix and the further removed matrix (Häuselmann, H. J., Aydelotte M. B., Schumacher B. L., Kuettner K. E., Gitelis, S. H., and Thonar, E. J.-M. A. (1992) Matrix 12, 116-129). To study the metabolism of aggrecan in these two compartments, mature bovine articular chondrocytes in alginate beads were pulsed with [35S]sulfate for 30 min or 16 h on day 7 of culture and then chased in isotope-free medium for up to 21 days. At different times, the two matrix pools were separately isolated, and the 35S-proteoglycans quantified, purified, and characterized. Radiolabeled aggrecan molecules exhibited a very long average half-life in the beads (t1/2 = 95 days). In contrast, small non-aggregating proteoglycans, which made up approximately 4% of the 35S-proteoglycans synthesized, were rapidly lost from the beads (t1/2 = < 24 h). Approximately half the 35S-aggrecan subunits, representing mostly molecules which showed a delay in ability to form aggregates in the presence of exogenous hyaluronan and link protein, spent only a short time (t1/2 = 4 h) in the cell-associated matrix before moving into the further removed matrix. They exhibited a much longer average half-life in the beads than 35S-aggrecan molecules which became resident of the cell-associated matrix (t1/2 = > 95 days versus 15 days). Radiolabeled aggrecan subunits in the two matrix compartments had a similar average hydrodynamic size and polydispersity; importantly, the size of these molecules did not change during the chase period. Catabolism of 35S-aggrecan in the cell-associated matrix was the only significant contributor to the appearance in the medium of partially degraded 35S-aggrecan which had lost the ability to bind to hyaluronan. These results strongly suggest aggrecan molecules which reside in the pericellular and territorial matrix compartments in close proximity to the chondrocytes have a much faster rate of turnover than their counterpart in the interterritorial areas further removed from the cells.