Previous analyses of p53 in 40 HLA‐A*0201(HLA‐A2)⁺ squamous cell carcinomas of the head and neck (SCCHN) indicated that 6/13 p53 missense mutations that were detected, S149C, T150R, V157F, Y220C, Y220H and E271K, occurred within HLA‐A2‐restricted cytotoxic T lymphocyte (CTL)‐defined p53 epitopes. Of the 6, the p53 S149C, Y220C and Y220H peptides were immunogenic. Anti‐p53 mutant S149C and Y220H effector cells cross‐reacted against the parental wild type sequence (wt) p53 peptides, whereas anti‐p53 Y220C effector cells were specific for the mutant peptide, p53 Y220C cDNA‐transfected HLA‐A2⁺ SaOS cells, and an HLA‐A2⁺ SCCHN cell line naturally expressing the mutation. These results indicate that the p53 Y220C mutation can be processed and presented for CD8⁺ T cell recognition. Furthermore, using an autologous PBMC/tumor system, anti‐p53 Y220C peptide‐effector cells recognizing the autologous tumor could also be generated. Our analysis of p53 in 10 additional HLA‐A2⁺ SCCHN tumors detected the p53 Y220C in 2/10 tumors raising the overall frequency of the p53 Y220C mutation to 6/50 (12%) HLA‐A2⁺ SCCHN tumors. In contrast, independent of their HLA class I genotypes, the p53 Y220C mutation frequency for all human tumors analyzed to date is ∼1.5%. This unexpectedly high frequency of the p53 Y220C mutation in HLA‐A2⁺ SCCHN suggests that vaccines targeting this mutation would not only be expected to induce robust anti‐tumor immune responses in HLA‐A2⁺ subjects, but also be more widely applicable than previously envisioned for any given p53 missense mutation. © 2007 Wiley‐Liss, Inc.