Type X collagen has a very limited distribution during skeletal development in regions of hypertrophic cartilage destined for degradation. In solution assay, type X collagen is degraded to a 32-kDa cleavage product which is resistant to further degradation, suggesting this product may have a function in skeletal development. In this study, we have identified the 32-kDa cleavage product of type X collagen present in the conditioned media (CM) during incubation of isolated 12-day chick tibiae in the absence of serum. In this culture system, chondrocytes throughout the tibial cartilages hypertrophied and deposited type X collagen within their matrix. During culture, the cartilage matrix was degraded in two stages. First proteoglycan was lost followed by degradation of the collagenous components. Collagen degradation was accompanied by the release of active interstitial collagenase and gelatinase into the CM. Purified type X collagen incubated in this CM was cleaved to form a 32-kDa product which was resistant to further degradation. This cleavage product has the same electrophoretic mobility as the 32-kDa chain produced by purified human collagenase.