Sub-populations of bovine articular chondrocytes derived from different depths of the cartilage showed differences in accumulation of proteoglycan-rich extracellular matrix in culture. To extend these morphological studies, the synthesis and catabolism of ³⁵S-labeled proteoglycans have been examined in similar cultures. Chondrocytes from deep zones synthesized significantly more proteoglycans than cells from the superficial zone. While all populations of chondrocytes synthesized pre-dominantly aggregating proteoglycans, a higher proportion of isotope was present in non-aggregating proteoglycans in cultures of superficial chondrocytes, by comparison with those of deep cells. Proteoglycans were degraded more rapidly by superficial cells than by chondrocytes from deeper layers. These results correlate both with previous histochemical studies of similar cultures, and with known depth-related variations in biochemical composition of intact articular cartilage.