The study of tissue-derived Mycobacterium leprae provides insights to the immunopathology of leprosy and helps identify broad molecular features necessary for mycobacterial parasitism. A major membrane protein (MMP-II) of in vivo-derived M. leprae previously recognized (Hunter, S.W., B. Rivoire, V. Mehra, B.R. Bloom, and P.J. Brennan. 1990. J. Biol. Chem. 265:14065) was purified from extracts of the organism and partial amino acid sequence obtained. This information allowed recognition, within one of the cosmids that encompass the entire M. leprae genome, of a complete gene, bfr, encoding a protein of subunit size 18.2 kD. The amino acid sequence deduced from the major membrane protein II (MMP-II) gene revealed considerable homology to several bacterioferritins. Analysis of the native protein demonstrated the iron content, absorption spectrum, and large native molecular mass (380 kD) of several known bacterioferritins. The ferroxidase-center residues typical of ferritins were conserved in the M. leprae product. Oligonucleotides derived from the amino acid sequence of M. leprae bacterioferritin enabled amplification of much of the MMP-II gene and the detection of homologous sequences in Mycobacterium paratuberculosis, Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium intracellulare, and Mycobacterium scrofulaceum. The role of this iron-rich protein in the virulence of M. leprae is discussed.