Elevated levels of the p53 protein occur in ∼50% of human malignancies, which makes it an excellent target for a broad-spectrum T cell immunotherapy of cancer. A major barrier to the design of p53-specific immunotherapeutics and vaccines, however, is the possibility that T cells may be tolerant of antigens derived from wild-type p53 due to its low level of expression in normal thymus and lymphohemopoetic cells. The combination of p53 deficient (p53^−/−) and p53^+/+ HLA-A2.1/K^b transgenic mice was used as a model to explore the possibility that A2.1restricted cytotoxic T lymphocytes (CTL) are functionally tolerant of self peptides derived from the wild-type p53 tumor suppressor protein. A2.1-restricted CTL specific for a naturally processed p53 self-epitope spanning residues 187-197 were completely aborted in p53^+/+ as opposed to p53^−/− transgenic mice. In contrast, CTL specific for a second self-epitope spanning residues 261-269 of the murine p53 sequence were detected in both p53^−/− and p53^+/+ A2.1/K^b transgenic mice. However, the avidity of the CTL effectors obtained from p53^+/+ mice was 10-fold lower than that obtained from p53^−/− mice, again suggesting elimination of CTL with high avidity for the A2.1-peptide complex. The circumvention of functional tolerance of high avidity CTL may therefore be a necessary prerequisite for optimizing immunotherapy against A2.1-restricted wild-type p53 epitopes in humans.