Accumulated evidence suggests that in the growth cartilage during endochondral ossification, proteoglycans in the extracellular matrix of the lower hypertrophic zone are degraded by proteases and removed before mineralization. Neutral protease activity in the human growth cartilage was demonstrated recently, with the highest levels in the hypertrophic and calcified zones. In this study, in an in vivo model, proteoglycans in the epiphyseal cartilage are cleaved at specific sites in the protein core by enzymes acting similar to neutral metallo-proteases. Prelabeled growth cartilage (35S) chondrocytes, grown in vitro, were transplanted as an allograft to fill a defect in the proximal tibial plate of immature New Zealand white rabbits and then extracted at different time intervals. Degradation of these proteoglycans was determined by gel chromatography on Sepharose 2B columns under associative and dissociative condition. This was compared to in vivo degradation patterns of flash-labeled native growth cartilage proteoglycan. Both the in vivo labeled material and the in vitro labeled transplants appear to be degraded into smaller fragments over time. This study provides further proof that the degradation of proteoglycans does occur in vivo and that this process is an important element in the preparation of bone for mineralization. Control of degradation may alter growth processes.