MHC class Ib molecule H2-M3 presents N-formylated peptides to CD8+ CTLs. Endogenous formylated peptides can come from the N-terminus of each of the 13 proteins encoded by the mitochondrial genome. In peptide competition assays, two of these peptides bind with high affinity, six bind with intermediate affinity, three bind with low affinity, and two do not bind measurably. Alloreactive CTLs from M3-specific, mixed lymphocyte cultures responded strongly against the two peptides with high affinity for M3, occasionally to peptides with intermediate affinity, and not at all to the rest. Long term lines and CTL clones reacted with only the high affinity peptides, demonstrating that alloreactive CTLs depend on specific peptides and that peptide affinity for class I correlates with alloantigenicity.