Recently, we observed that CTL specific for the wild-type (wt) sequence p53(264-272) peptide could only be expanded ex vivo from PBMC of a subset of the HLA-A2.1(+) normal donors or cancer patients tested. Surprisingly, the tumors of the responsive patients expressed normal levels of wt p53 and could be considered unlikely to present this epitope. In contrast, tumors of nonresponsive patients accumulated mutant p53 and were more likely to present this epitope. We sought to increase the responsive rate to the wt p53(264-272) peptide of PBMC obtained from normal donors and patients by identifying more immunogenic variants of this peptide. Two such variants were generated by amino acid exchanges at positions 6 (6T) and 7 (7W) of the peptide. These variants were capable of inducing T cells from PBMC of nonresponsive donors that recognized the parental peptide either pulsed onto target cells or naturally presented by tumors. TCR Vbeta analysis of two T cell lines isolated from bulk populations of effectors reactive against the wt p53(264-272) peptide, using either the parental or the 7W variant peptide, indicated that these T cells were expressing identical TCR Vbeta13.6/complementarity-determining region 3/J region sequences. This finding confirms the heteroclitic nature of at least one of the variant peptides identified in this study. The use of variant peptides of the wt p53(264-272) epitope represents a promising approach to overcoming the nonresponsiveness of certain cancer patients to this self epitope, thereby enhancing its potential use in tumor vaccines for appropriately selected cancer patients.