When 160 γg/ml sodium salicylate was added to the culture medium, glycosaminoglycan synthesis in slices of normal articular cartilage from habitually loaded areas of canine femoral condyles was diminished by 24% (P ≤ 0.01). Although glycosaminoglycan synthesis in cartilage from habitually unloaded regions of the same joints was similar to that in cartilage from loaded sites, the mean uronic acid content of the former was about 25% less, and salicylate suppressed glycosaminoglycan synthesis in unloaded cartilage to a much greater extent (42% of control) than it did in loaded cartilage (P ≤ 0.01). Furthermore, 1.5 γg/ml indomethacin, which had no effect on cartilage from loaded regions, suppressed glycosaminoglycan synthesis in cartilage from unloaded regions by 31%. However, if cartilage from loaded regions was treated with testicular hyaluronidase, indomethacin inhibited glycosaminoglycan synthesis, and inhibition of glycosaminoglycan metabolism by salicylate in hyaluronidase-treated cartilage was greater than in untreated cartilage. The data suggest that the effects of nonsteroidal antiinflammatory drugs on glycosaminoglycan metabolism in articular cartilage are dependent on proteoglycan content of the extracellular matrix.