Proteases have been postulated to account for the progressive disappearance of matrix proteoglycans in osteoarthritic (OA) cartilage. The digestion of endogenous proteoglycans by neutral proteases in human OA cartilage homogenates has been measured and compared with that of normal age-matched controls. Cartilage was obtained from 16 patients at the time of knee arthroplasty and from 7 accident victims. Tissue blocks were cut from the tibial plateau; part was used for histologic grading of the severity of OA and part was homogenized for the quantification of neutral metallo- and serine protease activities, based on the release of digested products from endogenous proteoglycans. Total metalloprotease activity (latent plus active forms) was elevated 3- to 10-fold in all diseased cartilage. This elevation was already significant in mild disease, but was greatest in samples of moderate to severe disease. The active form of the enzyme was highest at the center of erosions and decreased in the margins of the plateau. The digestion of proteoglycans, as distinct from their mere release from the tissue, was demonstrated by chromatography on Sepharose-CL2B and by large pore electrophoresis. Serine protease activity on proteoglycans was much lower than that of metalloprotease. The mean activity was highest in mild disease and declined in the severe disease samples, but the difference between these 2 groups and the controls was not statistically significant. The results of this study are consistent with the hypothesis that the neutral metalloproteases of cartilage are involved in the degradation of proteoglycans in osteoarthritis.