P53 is an attractive target immunotherapy because it is overexpressed in up to one half of all malignancies, and its overexpression often correlates with a worsened prognosis. We wanted to determine the feasibility of targeting wild-type epitopes p53 on human tumor cells. HLA A2.1 transgenic mice were immunized with the immunodominant wild-type p53 peptide epitopes, p53(149-157) and p53(264-272), along with a pan-DR helper epitope peptide in incomplete Freund's adjuvant (IFA). Twelve days later, splenocytes were harvested and stimulated with syngeneic blast cells that had been acid-treated to remove endogenous peptide and p53 peptide-pulsed. The responding cells were subsequently restimulated weekly with acid washed, peptide-pulsed Jurkat cells transfected with HLA A2.1. Peptide specific activity was tested in a chromium release assay. The resulting cytotoxic T cells (CTL) were cloned by limiting dilution. Peptide specific CTL were generated against both p53(149-157) and p53(264-272. Only p53(149-157) specific CTL were able to recognize and lyse cells that overexpressed endogenous p53. CTL clones derived from the p53(149-157) cell line demonstrated high affinity and specificity for p53(149-157) when presented by HLA A2.1+ cells. The p53(149-157) specific CTL were tested for specificity against a variety of cultured human cell lines. The CTL clones only lysed cells that overexpressed p53 in the context of HLA A2.1 and did not lyse cells with normal p53 expression or cells that lacked HLA A2.1 expression. This study demonstrates the possibility of targeting tumors, which overexpress p53, and raises the possibility transferring the high affinity, p53 specific T cell receptors from the murine CTL to human T cells.