In the present study, we show that a singly substituted peptide derived from the epitope MART1_27–35 and containing a Leu in position 1 (LAGIGILTV; 1L) behaves as a superagonist by in vitro inducing specific T cells with enhanced immunological functions. 1L-specific CTLs can be raised from peripheral blood of HLA-A2⁺ melanoma patients more efficiently than T cells specific for the cognate peptide. These T cells show a greater sensitivity to native MART1_27–35 when compared with CTL variable raised to parental peptide from the same patients. More importantly, anti-1L but not anti-native T cells display high levels of interferon γ production at early time points, and readily secreted interleukin-2 in response to native epitope endogenously presented by melanoma cells. Additionally, anti-1L T cells are insensitive to the inhibitory effects of MART1_27–35 natural analogues that antagonize the lytic response of CTLs raised to the cognate peptide. Analysis of T-cell receptor variable β usage suggests that the native and 1L peptides stimulate different components of the MART1_27–35-reactive T cell population. These data provide rationale to the use of superagonist analogues of tumor antigens for inducing in vivo immunization potentially able to overcome tumor immune escape and mediate a more significant control of tumor growth.