The production of autoantibodies to citrullinated type II collagen and the citrullination of type II collagen were analyzed in rheumatoid arthritis. Autoantibodies to citrullinated type II collagen were detected in 78.5% of serum samples from 130 rheumatoid arthritis patients. Autoantibodies to native noncitrullinated type II collagen were detected in 14.6% of serum samples, all of which were positive for anti-citrullinated type II collagen antibodies. Serum samples were also positive for anti-citrullinated type II collagen antibodies in 1 of 31 systemic lupus erythematosus patients and 2 of 55 patients with osteoarthritis of the knee. In contrast, sera samples from 24 systemic sclerosis patients, 21 dermatomyositis/polymyositis patients, 21 ankylosing spondylitis patients, and 18 psoriatic arthritis patients were all negative for anti-citrullinated type II collagen antibodies. Anti-citrullinated type II collagen antibodies and fragments of citrullinated type II collagen were found in the synovial fluid obtained from affected knee joints of 15 rheumatoid arthritis patients. Moreover, anti-citrullinated type II collagen antibodies were isolated from the synovium of affected knee joints in 8 rheumatoid arthritis patients using antigen/antibody immunocomplex dissociation buffer but not by using standard buffers. These findings indicate that autoantibodies that react with citrullinated type II collagen are specifically produced and that immunocomplexes composed of fragments of citrullinated type II collagen and autoantibodies are deposited in the inflamed articular synovium in rheumatoid arthritis patients. Assaying for the presence of anti-citrullinated type II collagen antibodies may therefore be useful for diagnosing rheumatoid arthritis, and the deposition of these immunocomplexes in the articular synovium may be involved in pathogenesis.