Sera from patients with primary biliary cirrhosis exhibit variable autoantibody reactivity against mitochondria, the commonest antigen (designated M2) including three structures of approximate M.W. 70, 50 and 40 kD. The nature of these antigens has only recently been established; the 70 and 50 kD are the transacetylase E2 and component X, respectively, of the pyruvate dehydrogenase complex and are distinct polypeptides. We have demonstrated, by immunoblotting, elution and rebinding of antibodies, unequivocal cross-reactivity between the major bands of the M2 antigen. In addition, cross-reactivity has been shown between antibodies binding to each of the three M2 bands of mitochondria and two major antigenic bands of both Gram-negative and Gram-positive bacteria. Conversely, antibodies eluted from these two bands of Escherichia coli were found to bind all three M2 bands of mitochondria. These results suggest that the antibodies of primary biliary cirrhosis contain both peptide-specific and cross-reacting antibodies, the latter recognizing a common “M2 epitope” that might include nonprotein components of the peptides. However, direct and competitive enzyme-linked immunosorbent assays failed to implicate the coenzyme of the pyruvate dehydrogenase complex, lipoic acid or its amide, as the common antigenic moiety.