Abstract Degradation of type II collagen is a central process in cartilage destruction seen in osteoarthritis and rheumatoid arthritis. Primary cleavage of type II collagen at the collagenase site is rate-limiting and is, therefore, a critical step for its degradation. The major contributor to this cleavage was identified in three isozymes of collagenase in human cartilage. Primary cultured human chondrocytes were used for the study. The production of collagenase-1 was major in total production for three isozymes of collagenase after stimulations with any concentration of tumor necrosis factor-α and/or interleukin-1 at 48 and 72 h, comprising 98% or greater of the total collagenase. When the production of collagenase-1 was specifically suppressed by the transfection with duplexes of 21-nucleotide small interfering ribonucleic acid into the cells, the activity of type II collagen cleavage was linearly decreased at neutral pH after activation. The relative contribution of collagenase-1 to the primary cleavage of type II collagen was determined to be 85% 93%. These findings suggest that collagenase-1 is a major contributor to the primary cleavage of type II collagens in human cartilage and is a potential therapeutic target for osteoarthritis and rheumatoid arthritis.