Low-affinity MHC class I-associated cryptic epitopes derived from self proteins overexpressed in a wide variety of human tumors or derived from antigens of viruses exhibiting a high mutation rate, could be interesting candidates for tumor and virus immunotherapy, respectively. However, identification of low-affinity MHC-associated epitopes comes up against their poor immunogenicity. Here we describe an approach that enhances immunogenicity of nonimmunogenic low-affinity HLA-A2.1-binding peptides. It consists of modifying their sequence by introducing a tyrosine in the first position (P1Y). P1Y substitution enhances affinity of HLA-A2.1-associated peptides without altering their antigenic specificity. In fact, P1Y variants of ten nonimmunogenic low-affinity peptides exhibited a 2.3- to 55-fold higher binding affinity and/or stabilized the HLA-A2.1 for at least 2 h more than the corresponding native peptides. More importantly, P1Y variants efficiently triggered in vivo native peptide-specific CTL which also recognized the corresponding naturally processed epitope. The possibility for generating CTL against any low-affinity HLA-A2.1-associated peptide provides us with the necessary tool for the identification of cryptic tumor and virus epitopes which could be used for peptide-based immunotherapy.