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We tested the hypotheses that Epstein-Barr virus (EBV) DNA levels in peripheral blood leukocytes (PBL) of transplant recipients with posttransplant lymphoproliferative disease (PTLD) (1) exceed those of patients without PTLD, (2) rise with or before clinical detection of the disease, and (3) fall with effective therapy. Using the polymerase chain reaction (PCR) and an endpoint dilution technique, we compared EBV DNA levels in sequential specimens from 5 patients with PTLD, 16 solid organ transplant recipients without PTLD, and 5 young adults with primary infectious mononucleosis (IM), and in single specimens from 21 healthy seropositive subjects. EBV DNA levels in the first two groups rose with induction of immunosuppression despite prophylactic acyclovir. Markedly elevated levels of EBV DNA were seen in 4 of 5 patients with PTLD at or before clinical diagnosis. The peak levels in these patients exceeded those of transplant recipients without PTLD (P = 0.02) and healthy adults with EM (P = 0.02). EBV DNA levels fell dramatically with effective therapy. Four of 21 healthy seropositive subjects demonstrated low levels of EBV DNA, similar to levels seen late in the course of patients with IM. We conclude that a semiquantitative PCR assay for EBV DNA in PBL can assist in the detection of PTLD and in monitoring the effect of therapy.