Suboptimal activation of T lymphocytes by tumor cells may contribute to the failure of the immune system to control tumor growth. We recently demonstrated that Melan-A/MART-1-reactive CTLs can be anergized by peptide analogues with partial agonist/antagonist functions, which selectively impair interleukin (IL)-2 release. Here we analyze the potential expression of partial agonist/antagonist peptides by tumor cells and their role in suboptimal T-cell activation. HLA-bound peptide fractions were eluted from HLA-A*0201/Melan-A/MART-1(+) melanoma cells and analyzed for reconstitution of the MART-1-specific T-cell epitope. Among the peptide fractions able to induce IFN-gamma release by MART-1-specific T cells, only fraction 43-44 activated IL-2 production by anti-MART-1 T cells, whereas the remaining two fractions acted as peptide antagonists by inhibiting IL-2 release in response to the native epitope. A comparable down-modulation of IL-2 release could also be induced by the MART-1-derived peptide 32-40, previously identified in one of the two anergizing fractions. A substantial deficit in IL-2 release was additionally detected in tumor-specific CD8(+) T cells infiltrating melanoma lesions. To overcome IL-2 impairment by peptide antagonists, anti-MART-1 T cells were generated by in vitro sensitization with the two optimized analogues Melan-A/MART-1(27-35) 1L (with superagonist features) and Melan-A/MART-1(26-35) 2L (with improved HLA-A*0201 binding). T cells raised with the superagonist Melan-A/MART-1(27-35) 1L showed resistance to the inhibition of IL-2 release mediated by melanoma-derived peptide fractions, whereas Melan-A/MART-1(26-35) 2L-specific T cells appeared to be as sensitive as T cells raised with the parental epitope. This resistance was associated with the enhanced ability of Melan-A/MART-1(27-35) 1L-specific T cells to release IL-2. Taken together, these data indicate that melanoma cells can process and present on their surface peptides inhibiting optimal T-cell activation against immunodominant epitopes and that the usage of optimized peptide analogues could represent a promising approach for overcoming tumor-induced immunosuppression and possibly designing more successful vaccines for cancer patients.