The target antigen recognized by H-2Kd-restricted cytotoxic T lymphocytes (CTLs) specific for a mutagen-induced antigen on DBA/2-derived tumor P815 was identified 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-2Kd 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 approximately 100 copies per tumor cell.