To treat patients with refractory cytomegalovirus (CMV) reactivation after allogeneic stem cell transplantation, a phase I/II clinical study on adoptive transfer of in vitro-generated donor-derived or patient-derived CMV pp65-specific CD8+ T-cell lines was performed. Peripheral blood mononuclear cells from CMV seropositive donors or patients were stimulated with HLA-A*0201-restricted and/or HLA-B*0702-restricted CMV pp65 peptides (NLV/TPR) and 1 day after stimulation interferon-γ)-producing cells were enriched using the CliniMACS Cytokine Capture System (interferon-γ), and cultured with autologous feeders and low-dose interluekin-2. After 7–14 days of culture, quality controls were performed and the CMV-specific T-cell lines were administered or cryopreserved. The T-cell lines generated contained 0.6–17×106 cells, comprising 54%–96% CMV pp65-specific CD8+ T cells, and showed CMV-specific lysis of target cells. Fifteen CMV-specific T-cell lines were generated of which 8 were administered to patients with refractory CMV reactivation. After administration, no acute adverse events and no graft versus host disease were observed and CMV load disappeared. In several patients, a direct relation between administration of the T-cell line and the in vivo appearance of CMV pp65-specific T cells could be documented. In conclusion, administration of CMV pp65-specific CD8+ T-cell lines was found to be feasible and safe, and enduring efficacy of administered CMV pp65-specific CD8+ T-cell lines could be demonstrated.