The effect of vigorous exercise on the reversibility of canine knee cartilage atrophy produced by immobilization of the leg was studied. In comparison to cartilage from the contralateral control knees, cartilage from knees which had been immobilized in a cast for 6 weeks showed an increase in water content and decreases in thickness, Safranin O staining of the matrix, uronic acid content, and net proteoglycan synthesis. In addition, the ability of both newly synthesized (35S) and total tissue proteoglycans to interact with hyaluronic acid to form aggregates was diminished; this was apparently due to an abnormality in the hyaluronate-binding region of the core proteins. If the casts were removed and the animals were then allowed to ambulate ad libitum for 3 weeks, all of these changes were reversed. However, knee cartilage from 3 dogs which had been run daily on a treadmill (6 miles/day) for 3 weeks after removal of the casts exhibited continuing decreases in thickness, Safranin O staining, and uronic acid content (mean 31%), even though net proteoglycan synthesis was increased (mean 16%) in comparison to that in control cartilage from the contralateral (nonimmobilized) knee. Furthermore, the abnormality in both 35S— and total tissue proteoglycans which precluded their interaction with high molecular weight hyaluronic acid persisted. In this respect, the proteoglycans were indistinguishable from those obtained from knee cartilage immediately following 6 weeks in a cast.