Current evidence suggests that the optimal vaccines for cancer should incorporate tumor-specific cytotoxic as well as helper epitopes. Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines, which could combine multiple tumor epitopes defined by CD8(+) CTLs, as well as CD4(+) T-helper cells. To test this possibility, we generated anti-p53 CD4(+) T cells from peripheral blood obtained from an HLA-DRB1*0401(+) donor by in vitro stimulation with dendritic cells and recombinant human p53 protein. We identified the wt p53(110-124) peptide as a naturally presented epitope. In a series of ex vivo experiments, performed in an autologous human system, we then demonstrated the ability of anti-wt p53(110-124) CD4(+) T cells to enhance the generation and antitumor functions of CD8(+) effector cells. The results demonstrate the crucial role of T helper-defined epitopes in shaping the immune response to multiepitope cancer vaccines targeting p53. This model of tumor-specific CD8(+) and CD4(+) T-cell interactions suggests that future vaccination strategies targeting tumor cells should incorporate helper and cytotoxic T cell-defined epitopes.