We investigated whether in situ hybridization for EBV RNA on routine cardiac biopsies could be used as a predictive test for the development of posttransplant lymphoproliferative disorder (PTLD) in cardiac transplant recipients. We examined the sensitivity of the test by determining the frequency of EBV-positive cells in cardiac biopsy specimens from patients with a known history of PTLD. Biopsy specimens obtained during routine monitoring for rejection before or shortly after the diagnosis of PTLD from 10 pediatric heart transplant patients were examined. Four of 74 specimens (5.4%) demonstrated EBV-positive lymphocytes in the cardiac biopsy rejection infiltrates. The four positive specimens were obtained from 3 different patients, all before the diagnosis of PTLD. Given the low number of cardiac biopsy specimens with EBV-positive lymphocytes, as well as the low incidence of PTLD in cardiac transplant patients, we conclude that a routine screening of all cardiac biopsy specimens using in situ hybridization for EBV with the intention of predicting PTLD is not warranted. However, in situ hybridization for EBV might be used in selected cases, such as those in which the transplant patient does not respond to immunosuppressive therapy for rejection. In these patients, the presence of EBV-positive lymphocytes in biopsy specimens initially interpreted as showing rejection might instead raise the suspicion of incipient PTLD.