The frequent detection of human cytomegalovirus (CMV) antigens in glioblastoma multiforme (GBM) has raised the possibility of exploiting CMV-specific T-cell immunotherapy to control this disease in CMV--seropositive patients. Here, we have conducted a comprehensive ex vivo profiling of CMV-specific CD8(+) T-cell responses in a cohort of GBM patients. Of the patients analyzed, approximately half exhibited serological evidence of past infection with CMV. Although no CMV-specific CD8(+) T-cell responses could be detected in the serologically negative GBM patients, virus-specific CD8(+) T-cell responses were detected in all seropositive GBM patients. Using major histocompatibility complex-peptide multimers, the frequency of CMV-specific T-cells in the patients detected ranged from 0.1 to 22% of CD8(+) T-cells and a high proportion of these cells were positive for the human natural killer-1 glycoprotein CD57. Furthermore, ex vivo polychromatic functional analysis of the CMV-specific T-cells from GBM patients revealed that large proportions of these cells were unable to produce multiple cytokines (macrophage inflammatory protein (MIP)-1β, tumor necrosis factor (TNF)α and interferon (IFN)γ) and displayed limited cytolytic function (CD107a mobilization) following stimulation with CMV peptide epitopes. However, in vitro stimulation with CMV peptide epitopes in the presence of γC cytokine dramatically reversed the polyfunctional profile of these antigen-specific T-cells with high levels of MIP-1β, TNFα, IFNγ and CD107a mobilization. Most importantly, adoptive transfer of these in vitro-expanded T-cells in combination with temozolomide (TMZ) therapy into a patient with recurrent GBM was coincident with a long-term disease-free survival. These studies provide an important platform for a formal assessment of combination therapies based on CMV-specific T-cells and TMZ for recurrent GBM.