O ne approach to enhancing the T cell response to tumors is vaccination with mimotopes, mimics of tumor epitopes. While mimotopes can stimulate proliferation of T cells that recognize tumor-associated antigens (TAAs), this expansion does not always correlate with control of tumor growth. We hypothesized that vaccination with mimotopes of optimal affinity in this interaction will improve antitumor immunity. Using a combinatorial peptide library and a cytotoxic T lymphocyte clone that recognizes a TAA, we identified a panel of mimotopes that, when complexed with MHC, bound the TAA-specific TCR with a range of affinities. As expected, in vitro assays showed that the affinity of the TCR-peptide-MHC (TCR-pMHC) interaction correlated with activity of the T cell clone. However, only vaccination with mimotopes in the intermediate-affinity range elicited functional T cells and provided protection against tumor growth in vivo. Vaccination with mimotopes with the highest-affinity TCR-pMHC interactions elicited TAA-specific T cells to the tumor, but did not control tumor growth at any of the peptide concentrations tested. Further analysis of these T cells showed functional defects in response to the TAA. Thus, stimulation of an antitumor response by mimotopes may be optimal with peptides that increase but do not maximize the affinity of the TCR-pMHC interaction.