A human mitochondrial protein, designated P1 (63 kilodaltons [kDa], shows extensive sequence homology (47% identical residues and an additional approximately 20% conserved changes) to the 65-kDa mycobacterial antigen. To understand the relationship of these proteins, the cross-reactivity of several monoclonal antibodies directed against the 65-kDa Mycobacterium leprae antigen towards human, Chinese hamster, chicken, and bacterial cells has been examined. A number of antibodies (Y1-2, ML 30-A2, and F47-9-1) were found to cross-react with a 63-kDa antigen in vertebrate cell extracts and stained mitochondria in immunofluorescence studies. Some of these antibodies also reacted with a P1-beta-galactosidase fusion protein in recombinant Escherichia coli cells, expressing part of the human P1 protein. These results provide strong evidence that P1 is the mammalian homolog of the 65-kDa antigen. The human P1 protein also shows significant similarity (P less than 0.001) to a number of other bacterial and viral proteins including the pol polyprotein of human immunodeficiency viruses and the penicillin-binding protein of Neisseria gonorrhoeae. The observed similarity between human P1 protein and the major antigenic proteins of pathogenic organisms (e.g., 60- to 65-kDa mycobacterial antigen) suggests its possible involvement in autoimmune diseases (e.g., rheumatoid arthritis) by antigenic mimicry.