We determined the prevalence and antigenic specificity of autoantibodies against cytoskeletal proteins in patients affected with various autoimmune diseases. Sera collected from patients with rheumatoid arthritis, systemic lupus erythematosus or progressive systemic sclerosis, and normal volunteers, were examined for the presence of autoantibodies against cytoskeletal proteins by indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Patients with rheumatoid arthritis had the highest reactivity to cytoskeletal antigens on immunofluorescence assays using isolated muscle myofibrils (41/50) and L929 cells (37/50). Antigen-specific ELISA revealed significant immunoreactivity against actin (11/50) and myosin (15/50). In nine patients, immunoreactivity was seen against multiple cytoskeletal antigens. We concluded that the prevalence of IgG autoantibodies against cytoskeletal antigens, especially myofibrillar components actin and myosin, is elevated in patients with rheumatoid arthritis.