Previous studies have shown that substitution of single amino acid residues in human Melan-A immunodominant peptides Melan-A 27–35 and Melan-A 26–35 greatly improved their binding and the stability of peptide/HLA-A* 0201 complexes. In particular, one Melan-A peptide analogue was more efficient in the generation of Melan-A peptide-specific and melanoma-reactive CTL than its parental peptide in vitro from human PBL. In this study, we analyzed the in vivo immunogenicity of Melan-A natural peptides and their analogues in HLA-A* 0201/K b transgenic mice. We found that two human Melan-A natural peptides, Melan-A 26–35 and Melan-A 27–35, were relatively weak immunogens, whereas several Melan-A peptide analogues were potent immunogens for in vivo CTL priming. In addition, induced Melan-A peptide-specific mouse CTL cross-recognized natural Melan-A peptides and their analogues. More interestingly, these mouse CTL were also able to lyse human melanoma cell lines in vitro in a HLA-A* 0201-restricted, Melan-A-specific manner. Our results indicate that the HLA-A* 0201/K b transgenic mouse is a useful animal model to perform preclinical testing of potential cancer vaccines, and that Melan-A peptide analogues are attractive candidates for melanoma immunotherapy.