T-cell immunotherapy that takes advantage of Epstein-Barr virus (EBV)–stimulated immunity has the potential to fill an important niche in targeted therapy for EBV-related cancers. To address questions of long-term efficacy, safety, and practicality, we studied 114 patients who had received infusions of EBV-specific cytotoxic T lymphocytes (CTLs) at 3 different centers to prevent or treat EBV⁺ lymphoproliferative disease (LPD) arising after hematopoietic stem cell transplantation. Toxicity was minimal, consisting mainly of localized swelling at sites of responsive disease. None of the 101 patients who received CTL prophylaxis developed EBV⁺ LPD, whereas 11 of 13 patients treated with CTLs for biopsy-proven or probable LPD achieved sustained complete remissions. The gene-marking component of this study enabled us to demonstrate the persistence of functional CTLs for up to 9 years. A preliminary analysis indicated that a patient-specific CTL line can be manufactured, tested, and infused for $6095, a cost that compares favorably with other modalities used in the treatment of LPD. We conclude that the CTL lines described here provide safe and effective prophylaxis or treatment for lymphoproliferative disease in transplantation recipients, and the manufacturing methodology is robust and can be transferred readily from one institution to another without loss of reproducibility. The current trial was registered at www.clinicaltrials.gov as #NCT00058812.