OBJECTIVE Different infectious agents have been proposed to be involved in the pathogenesis of rheumatoid arthritis (RA). We investigated the role of latent Epstein-Barr virus (EBV) infection in patients with RA. METHODS Sera of 55 patients with RA and 60 age and sex matched healthy controls were examined for antibodies against EBV encoded antigens (EBNA-1, VCA, and EA) by ELISA. The presence of viral DNA was analyzed by polymerase chain amplification of DNA isolated from peripheral blood mononuclear cells (PBMC) of these samples and from cells of synovial fluid (SF) specimens. Paraffin sections of synovial membranes from 25 patients were subjected to in situ hybridization analysis for the EBV encoded small RNA EBER1 and EBER2. RESULTS Two-fold increased values of IgG antibodies against EBNA-1 were found in patients with RA in comparison to healthy controls (p = 0.029). No statistically significant difference could be observed for antibody levels against EBV-VCA. Fourteen (24.1%) of 55 patients with RA had serological evidence of reactivated EBV infection in comparison to none of the control group (p = 0.028). In PBMC, EBV DNA was detected in a significantly higher proportion in the patient group (50.9 vs. 30%; p = 0.02). In addition, SF cells harbored the viral DNA in 30% of RA cases compared to 16.6% of control cases (p = 0.02). However, EBER1/2 transcripts could only be found within synovial membranes of 2 (8%) of 25 patients with RA. CONCLUSIONS These findings support the hypothesis that EBV infection may be involved in the pathogenesis of RA. Further studies may define the precise pathogenetic mechanisms of viral infection for the development of inflammatory arthritis.