Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease is a life-threatening complication following hematopoietic stem cell transplantation. A quantitative polymerase chain reaction to evaluate EBV-genome copy numbers based on a nested polymerase chain reaction and an end-point dilution was used. Applying this assay EBV load was prospectively screened weekly in 123 patients after transplantation. The results demonstrate that EBV reactivations with more than 1,000 EBV-genome copies measured in 105 peripheral blood mononuclear cells were observed in 31 patients (25.2%). Three patients developed lymphoproliferative disease with extremely high EBV-genome copies in peripheral blood mononuclear cells (>100,000 copies/105 cells) and plasma. After combined antiviral and immune therapy two of three patients showed a dramatic decrease of EBV load and survived, while the third patient died of lymphoma. A subclinical EBV reactivation was observed in 24 cases (19.5%) with EBV-genome copies in 105 peripheral blood mononuclear cells ranging between 2,500 and mostly 10,000. After reduction of immunosuppression the EBV levels normalized. In four patients, the high copy number of ≥80,000 copies/105 peripheral blood mononuclear cells and plasma positivity prompted us to start pre-emptive therapy with rituximab and cidofovir for prevention of lymphoproliferative disease. After drug administration the high EBV load was reduced remarkably. Ninety-two patients (74.8%) who had ≤1,000 copies/105 peripheral blood mononuclear cells did not develop EBV-associated lymphoproliferative disease. In conclusion, monitoring of EBV load is a sensitive and useful parameter in the surveillance of EBV reactivation for early intervention in EBV-associated lymphoproliferative disease as well as for follow-up of the efficacy of therapy.