Peptide-based vaccines are currently being tested for their ability to induce or augment tumor antigen (Ag)-specific CD8+ T-cell responses in cancer patients. Here we report that the frequency of circulating CD8+ T cells directed against the Melan-A/MART-1 Ag increased >20-fold in an HLA-A2 melanoma patient immunized repeatedly with the corresponding antigenic peptide, as assessed by staining with HLA-A2/peptide tetramers. Multiparameter flow cytometric analysis demonstrated that the increase in total Melan-A-specific cell number was accompanied by a marked increase in the proportion of the cells that expressed an activated/memory surface phenotype. As assessed by ELISPOT assays and intracellular staining, the absolute number of Melan-A-specific cells able to secrete IFN-gamma increased >50-fold upon vaccination. When tested directly after cell sorting on the basis of tetramer staining, Melan-A-specific cells were weakly cytolytic but became highly active after in vitro restimulation. Altogether, these results indicate that large numbers of functionally active tumor Ag-specific CD8+ T cells can be obtained and maintained at high levels after in vivo activation by repeated peptide-based vaccination.