We have studied a case of human primary melanoma displaying the classical signs of a spontaneous regression in order to characterize potentially efficient anti-tumor T cell responses. In a previous series of experiments a unique TCR Vbeta16+ T cell was shown to be highly expanded at the tumor site. The corresponding clone was isolated in vitro and found to be a CD8+ cytotoxic T lymphocyte with a strong and selective cytolytic activity against the autologous tumor cell line. Here, we demonstrate that this predominant Vbeta16+ tumor-infiltrating lymphocyte recognizes a peptide encoded by a novel unconventional myosin class I gene. This peptide includes a mutation due to a single nucleotide substitution. The resulting Glu-->Lys replacement at position 911 of the coding sequence is critical to generate the recognized T cell epitope. These experiments demonstrate the existence of a natural tumor-specific cytolytic T cell response in a primary regressing human melanoma lesion.