Thirty-five HLA-A2⁺ patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100_209–2M, emulsified in Montanide adjuvant. Direct ex vivo gp100_209–2M tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer⁺ CD8⁺ T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05–8.9%). Ex vivo IFN-γ cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100_209–2M in vitro activation showed that for all of the patients studied tetramer⁺ CD8⁺ T cells produced IFN-γ; however, some patients had significant numbers of tetramer⁺ IFN-γ⁻ CD8⁺T cells suggesting functional anergy. Additionally, 8 day gp100_209–2M in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer⁺ CD8⁺ T cells from postvaccine cells for 34 patients, and these IVS tetramer⁺ CD8⁺ T cells were functionally responsive by IFN-γ cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8⁺ T cells demonstrated the proliferation of functionally anergic gp100_209–2M- tetramer⁺ CD8⁺ T cells in several patients and also indicated interpatient variability of gp100_209–2M stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer⁺ CD8+ T cells (78.1 ± 4.2%) had either an “effector” (CD45 RA⁺/CCR7⁻) or an “effector-memory” phenotype (CD45RA⁻/CCR7⁻). Notably, analysis of PBMCs collected 12–24 months after vaccine therapy demonstrated the durable presence of gp100_209–2M-specific memory CD8⁺ T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8⁺ T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.