&NA;In this study we directly compared thein vitroresponses of T‐cells from normal donors and melanoma patients to Melan‐A27‐35and Melan‐A26‐35. These peptides have been previously used in peptide‐based vaccination studies. Following three stimulations with peptide‐pulsed antigenpresenting cellsin vitro,Melan‐A‐specific cytolytic T‐lym‐phocytes (CTLs) were generated from seven of 20 subjects; two of the seven subjects responded reproducibly to both Melan‐A27‐35and Melan‐A26‐35, three to only Melan‐A27‐35and two to only Melan‐A26‐35. However, CTLs generated with either Melan‐A27‐35or Melan‐A26‐35showed 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‐A27‐35specific CTLs, while others may favour Melan‐A26‐35specific 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‐A27‐35and Melan‐A26‐35peptides in clinical trials.