Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) are considered to be one of major defenses against the activation of EBV infection. We have developed a simple assay for evaluation of EBV-CTL by means of culturing peripheral blood mononuclear cells at the concentration of 2 x 10(6) cells/ml infected by B95-8 EBV, in the presence or absence of cyclosporin A (CSA). No lymphoblastoid cell line was established from ten EBV-seropositive healthy individuals in the absence of CSA. In contrast, cell lines were established from ten EBV-seronegative healthy individuals in the absence of CSA, indicating lack of EBV-CTL in these individuals. Methods described herein are simple and correlate well with EBV-CTL activity when compared to conventional methods, such as outgrowth regression or chromium-51 release assays.