The osteoarthritis (OA) process is characterized by the progressive destruction of articular cartilage. There is a loss of cartilage proteoglycan content and disorganization of the collagen network, as well as an increase in other non-collagenous protein such as fibronectin (Fn). Increased proteolytic activity may lead to the degradation of native Fn and generation of Fn proteolytic fragments. Among them, the 45 kDa collagen-binding Fn fragment can be autoactivated in vitro into a 40 kDa fragment. This 40 kDa fragment induces an average of 30% of proteoglycan release per day from human OA cartilage explants and can degrade proteoglycan using dead cartilage sections. Proteoglycan-degrading activity related to the 40 kDa Fn fragment was decreased up to 66% by fetal calf serum (10%), but was not prevented by protein synthesis inhibitors (cycloheximide or actinomycin D). The action of this 40 kDa Fn fragment was greater on OA than on normal cartilage. This study suggests that enzymatic activity induced by the 40 kDa collagen-binding fragment of Fn might be involved in cartilage matrix turnover.