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In this study we directly compared the in vitro responses of T-cells from normal donors and melanoma patients to Melan-A27-35 and Melan-A26-35. These peptides have been previously used in peptide-based vaccination studies. Following three stimulations with peptide-pulsed antigen-presenting cells in vitro, Melan-A-specific cytolytic T-lymphocytes (CTLs) were generated from seven of 20 subjects; two of the seven subjects responded reproducibly to both Melan-A27-35 and Melan-A26-35, three to only Melan-A27-35 and two to only Melan-A26-35. However, CTLs generated with either Melan-A27-35 or Melan-A26-35 showed cross recognition, and both types of CTL could recognize naturally processed antigen displayed on HLA-A2+ tumour cells. Furthermore, Melan-A-specific CTLs could also be generated by stimulating peripheral blood mononuclear cells with autologous melanoma cells. Our results suggest that some subjects may have a bias in their CTL repertoire which favours the generation of Melan-A27-35 specific CTLs, while others may favour Melan-A26-35 specific CTLs. It is also likely that CTL precursors capable of detecting both peptides may have different affinities to the two Melan-A peptides. Since it is difficult to predict the CTL responses to Melan-A peptide in a given individual, we suggest vaccinating with both Melan-A27-35 and Melan-A26-35 peptides in clinical trials.