OBJECTIVE To investigate the role of superantigen in rheumatoid arthritis (RA) by assaying the serum levels of staphylococcal enterotoxin B (SEB) antibodies. METHODS Serum IgG and IgM SEB antibodies were measured using an enzyme linked immunosorbent assay (ELISA), and confirmed by Western blot analysis. The T cell receptor V beta (TCR V beta) repertoire was analysed using the reverse transcriptase polymerase chain reaction. RESULTS RA patients had increased levels of serum IgM SEB antibody compared with normal subjects, patients with systemic lupus erythematosus, Sjögren's syndrome, and Behçet's disease. The titres of rheumatoid factor (RF) showed no correlation with the levels of IgM SEB antibodies, and the levels of SEB antibodies were not inhibited by the addition of human immunoglobulin, or after absorption of RF. RA patients whose disease duration was less than 10 years had greater levels of serum IgM SEB antibodies than those with disease duration more than 10 years. The levels of IgM and IgG SEB antibodies in synovial fluid from RA patients were correlated with those in their sera. Western blot analysis detected IgM and IgG SEB antibodies as a band of approximately 30 kDa molecular size. The percentage of TCR V beta 2, V beta 5.2, and V beta 12 in phytohaemagglutinin stimulated peripheral T cells correlated significantly with the levels of serum IgM SEB antibody in RA patients. CONCLUSIONS These results suggest that SEB, one of the superantigens, may have a critical role in the pathogenesis of RA.