The expression of 65-kd mycobacterial heat-shock protein (HSP)-related antigens in synovial membrane from rats and humans with arthritis was studied using three monoclonal antibodies and one polyclonal antiserum directed to antigens of mycobacteria. The antibodies labeled synovial tissue sections from both adjuvant arthritis (AA) rats and from patients with either rheumatoid arthritis (RA) or osteoarthritis (OA); especially the synovial lining cells appeared to be positive. The cytoplasmic staining patterns in rats and humans were essentially the same and were not related to the extent of inflammation ie, the size of lymphoid infiltration. In control tissues no cytoplasmic staining was observed. The results suggest a role for a 65-kd HSP or a cross-reactive molecule in the immunopathologic process of arthritic disease.