The rat maternally transmitted Ag has been previously described as a minor histocompatibility Ag composed of a mitochondrially transmitted factor (MTF) and the RT1.Aa MHC class I molecule. We compared the DNA sequences of the 13 mitochondrial open reading frames from different rat strains and identified four coding polymorphisms that correlated with this MTF. We used synthetic 17-mer peptides spanning the polymorphisms to sensitize appropriate target cells in lymphocytotoxicity assays and found that the MTF is derived from an internal region of ATPase 6. A tridecameric derivative of the ATPase 6 17 mer (termed 13N3E) could sensitize RT1.Aa-expressing target cells at picomolar concentrations and, when present on such cells, could compete fully with the natural ligand in cold-target competition assays. Comparing the 13N3E peptide with the known peptide-binding requirements of RT1.Aa suggested two possible binding conformations, placing either an internal or a C-terminal arginine in the F pocket of the peptide-binding groove. Arguments favoring a "bulging" conformation, with N- and C-terminal residues bound into their conserved pockets, are discussed.