Reactivity to the mycobacterial 65 kDa heat shock protein (HSP 65) has been implicated in the pathogenesis of adjuvant arthritis in the rat, and may be involved in the pathogenesis of rheumatoid arthritis or other autoimmune diseases in humans. Accordingly this study sought quantitative or qualitative differences in the antibody reactivity to HSP 65 between normal controls, patients with the multisystem autoimmune diseases, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and patients with the mycobacterial infections, tuberculosis (TB) and leprosy. Levels of antibodies to recombinant HSP 65 in serum were measured by ELISA in normal subjects and in patients with RA, SLE, TB or leprosy. Antibody reactivity was examined by Western blotting using polypeptide fragments of HSP 65 derived by recombinant DNA techniques, or by digestion with trypsin or cyanogen bromide (CNBr). Reactivity to a synthetic peptide, the adjuvant arthritis T-cell epitope of HSP 65 (180-188), was tested by ELISA. High levels of antibodies to full length recombinant HSP 65 from Mycobacterium bovis were present in all the groups tested. By Western blot analysis, most reactivity with intact HSP 65 was retained in a 32 kDa tryptic fragment, judged by sequencing and size estimations to represent amino acid residues 118- approximately 388. This sequence included a major T-cell epitope for adjuvant arthritis (180-188), but these nine amino acids were not essential for B-cell reactivity since most sera also reacted with residues 188-540 which lack the T-cell epitope. Moreover, the 180-188 synthetic peptide was unreactive by ELISA, and did not inhibit reactivity with the intact recombinant HSP 65. In conclusion, most individuals had antibodies to mycobacterial HSP 65, presumably resulting from previous bacterial infections. The magnitude of the response was unrelated to the occurrence of systemic autoimmune disease, and the pattern of antibody reactivity with recombinant and proteolytic fragments of HSP 65 suggests that the major B-cell epitope is conformational and consists of discontinuous regions of the molecule.