We report here on 2 patients who received adjuvant vaccination with an HLA-A2– or HLA-A24–restricted tyrosinase peptide, respectively, and GM-CSF for frequently relapsing stage IV melanoma. Following resection of metastases and irradiation of brain metastases in 1 patient, both patients were without evidence of disease when receiving the first vaccination. While the patients had had 9 and 12, respectively, mostly s.c., relapses during the 3 years before vaccination, they experienced freedom from relapse for more than 2 years after vaccination. We found a T-cell response to the vaccine peptide in both patients in the peripheral blood by ex vivo IFN-γ ELISPOT assay. The T-cell population could be further characterized by 4-color flow cytometry in 1 patient, showing that the majority of the peptide-specific CD3+CD8+IFN-γ+ T cells were granzyme B-positive and CCR-7-negative, characterizing them as effector T cells with the ability to mediate cytotoxicity and migrate to inflamed tissues. In this patient also, augmentation of the T-cell response to autologous tumor cells by vaccination could be detected. A single-site postvaccination relapse occurred in both patients, showing downregulation of tyrosinase expression in 1 patient, while normal expression levels for tyrosinase, MHC class I antigens and components of the antigen-processing machinery were found in the other patient. These results suggest that peptide vaccination resulted in a prolonged relapse-free interval in these high-risk patients. © 2002 Wiley-Liss, Inc.