Several investigators have demonstrated an association between Epstein-Barr virus (EBV) and the pathogenesis of rheumatoid arthritis (RA). However, there is no direct evidence that this virus exists in the synovial cells of patients with RA. We attempted to detect EBV in synovial cells from RA patients. Specimens of synovial tissues from 34 patients with RA and from 20 patients with osteoarthritis (OA), and from one patient with psoriatic arthritis as controls, were examined for evidence of the EBV by in situ hybridization. The specimens were also tested by immunoperoxidase staining for expression of the CD21 molecule (EBV receptor), EBV nuclear antigen (EBNA)-2 and latent membrane protein (LMP)-1. EBV-encoded small RNA-1 (EBER) was demonstrated in synovial lining cells from eight (23.5%) out of 34 RA patients but in none of 20 OA patients (P < 0.05) nor in the one psoriatic arthritis patient. Interestingly, EBER localized in synovial lining cells that were located at the apex of villus proliferating lesions. Furthermore, LMP-1 was also detected in synovial lining cells at the top of villus lesions. Nevertheless, CD19 and CD21 molecules, and EBNA-2 were not demonstrated in such lesions. The incidence of EBV-positive in synovial lining cells with severely infiltrated lymphocytes tended to be higher than that in moderately infiltrated ones. This is the first evidence that EBV exists in chronically inflamed synovial lining cells of human joints in RA.