We previously described a motif prediction of major histocompatibility complex allele-specific peptides and an in vitro assay for actual measurement of peptide binding to human leukocyte antigen HLA-A2.1 molecules. Using this method we have identified candidate cytotoxic T lymphocyte (CTL) epitopes derived from a non-self-protein (influenza matrix) and self-protein (p53). We now show that results of binding assays performed over a range of peptide concentrations indicate that distinct differences in HLA-A2.1 peptide binding affinities exist between the influenza matrix and p53 protein. The results for the influenza matrix protein indicate that the peptide that shows the highest binding affinity to HLA-A2.1 is identical to the known immunodominant peptide recognized by influenza virus-specific CTLs. The results for p53 indicate that one of the peptides with a low binding affinity is capable of inducing specific CTL responses, but CTLs recognizing the highest affinity binding peptides were not obtained. These findings are discussed in terms of the distinct implications for induction of cellular immune responses directed against peptides with different binding affinities for HLA-A2.1 of proteins that constitute attractive targets for tumor immunotherapy.