The in vivo cytotoxic mechanism of Epstein--Barr virus (EBV)-specific cytotoxic lymphocytes was examined in a patient who suffered with EBV-associated lymphoproliferative disease (LPD) after bone marrow transplantation (BMT). His peripheral CD8+ T-cell count was significantly increased and >70% of these cells were EBV-specific by fluorescence-activated cell sorter (FACS) analysis for interferon-gamma production. Intracellular perforin expression was markedly increased in CD8+ T cells by FACS analysis. The lymphocytes from this patient had cytotoxic activity against autologous EBV+ lymphoblastoid cell lines which were completely inhibited by concanamycin A, an inhibitor of perforin, and a anti-human leucocyte antigen (HLA)-class I monoclonal antibody. These results suggest that the cytotoxicity was mediated by the perforin, in an HLA-class I-restricted manner. We performed serial intracellular perforin analyses in another patient who also showed endogenous expansion of EBV-specific CD8+ T cells that coincided with an increased EBV-DNA load. Perforin expression in the CD8+ and CD4+ T cells paralleled the EBV-specific CD8+ T cells and EBV-DNA load, which also suggests that perforin mediates EBV-specific cytolysis in vivo and is responsible for effective immunosurveillance against EBV reactivation after BMT. Evaluation of host immunity against EBV by determining perforin expression in lymphocytes and EBV-specific lymphocytes along with quantification of EBV-DNA may be useful for predicting the clinical course of patients with EBV-associated LPD after BMT.