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.