The target antigen recognized by H-2K^d-restricted cytotoxic T lymphocytes (CTLs) specific for a mutagen-induced antigen on DBA/2-derived tumor P815 was indentified as the product of a normal cellular gene encompassing a point mutation. Using synthetic peptides, the epitope recognized by these CTLs was narrowed down to be contained within the undecamer KYQAVTTTLEE, Incorporating the point mutation. The allele-specific peptide motif for H-2K^d molecules allowed us to predict the peptide naturally presented by the tumor cells to be the nonamer KYQAVTTTL. Isolation of the natural tum⁻-specific peptide from P198.3 tumor cells and biochemical comparison with the synthetic nonamer confirmed the prediction. This natural nonapeptide is represented by ∼100 copies per tumor cell.