Reactivation of EBV (Epstein-Barr virus) after bone marrow transplantation can result in EBV-associated lymphoproliferative disease (EBV-LPD). We have administered donor-derived EBV-specific cytotoxic T lymphocytes (CTL) to patients who are at high risk of this complication after receiving a T-cell-depleted allograft from a matched unrelated or mismatched related donor. The cells were marked with the neo gene before infusion so that we could evaluate their persistence and efficacy. CTL infusion produced a virus-specific immune response to EBV that persisted for up to 2 years. None of the 36 patients who received prophylactic CTLs have developed EBV-LPD, compared with a cumulative risk of 14% in patients who did not receive this treatment. Strong evidence of clinically valuable immune activity comes from 6 of these 36 patients whose pre-CTL levels of EBV DNA were elevated to a degree strongly predictive of the onset of lymphoma. In each of these cases, the levels returned to baseline after CTL infusion. 2 patients who were treated for clinically evident EBV-LPD attained prolonged remission after CTL infusion and in situ hybridization and semiquantitative PCR showed that the gene-marked CTL had selectively accumulated at disease sites. The prophylactic CTL treatment lacked acute adverse effects, whereas 1 patient who received CTLs for bulky established disease developed initial tumor swelling and respiratory obstruction. We conclude that EBV-specific CTLs are a safe and effective prophylaxis for EBV lymphoma and can also eradicate established disease. This approach is now being extended to other viruses that produce post-transplant morbidity and to other EBV-associated malignancies.