MTF, a murine minor histocompatibility antigen, is maternally inherited and thought to be encoded by a mitochondrial gene. We sequenced the entire mitochondrial genomes from three strains that differ in MTF Mtf beta, Mtf gamma, and Mtf delta) and compared the sequences with the known, Mtf alpha, mitochondrial DNA sequence. We found only one site where all four genomes differed, affecting amino acid residue 6 of ND1, a subunit of NADH dehydrogenase. Incubation of non-Mtf alpha target cells with synthetic peptide ND1 alpha 1-17 (the first 17 amino acid of the ND1 protein of Mtf alpha mice) rendered them susceptible to lysis by MTF alpha-specific cytotoxic T cells (CTLs). Similarly, non-Mtf beta target cells were lysed by MTF beta-specific CTLs after incubation with the allelic form ND1 beta 1-17. Thus, Mtf is attributable to allelic variation at a single residue of the ND1 protein. Cells can therefore display peptides derived from mitochondrially encoded proteins, and such peptides can be histocompatibility antigens.