Although Rheumatoid Arthritis (RA) is categorized in the systemic autoimmune diseases, specific autoantibodies in RA have been reported to be rare. We have investigated autoantibodies found in sera from patients with RA and examined their clinical significance. Immunoprecipitation techniques using 32P-labeled or 35S-labeled HeLa cell extracts and immunoblotting were utilized to detect autoantibodies in sera from 42 patients with RA and 58 patients with other connective tissue diseases as controls. Certain autoantibodies were detected in 33 of 42 patients (79%) with RA by any of the assay systems mentioned above. Anti-SSA/Ro, anti-SSB/La, anti-7-2RNP, and anti-Sm antibodies were found in 13 (29%), 3 (7%), 4 (10%), and 1 (2%) patients with RA, respectively. All 3 patients with anti-SSB/La and 9 with anti-SSA/Ro revealed Sjögren's syndrome besides RA. However, all 4 patients with anti-7-2RNP and one patient with anti-Sm showed no evidence of scleroderma nor SLE. Besides of these known autoantibodies, we have also detected 4 new antibodies which reacted with a 60kD protein in 15 (36%), 45kD in 10 (24%), 75kD in 8 (19%), 180kD in 4 patients (10%) with RA. These autoantibodies were not found in other connective tissue diseases except one of SLE for anti-60kD and one of dermatomyositis for anti-75kD, and therefore appeared to be specific for RA patients. Anti-45kD antibodies were associated with low prevalence of anti-nuclear antibodies, and anti-180kD antibodies were associated with lymphadenopathy and Sjögren's syndrome. Thus, it was demonstrated that patients with RA develop a variety of autoantibodies as well as those with other connective tissue diseases.