The epithelial cell adhesion molecule is strongly expressed in carcinomas of diverse histologic origin and has attracted attention as a target for immunotherapy. We have previously demonstrated that the epithelial cell adhesion molecule-derived human leukocyte antigen-A*0201-restricted nonamer peptide ILYENNVIT184–192can be efficiently recognized by peptide-specific cytotoxic T lymphocytes (CTL). Using this peptide (Ep-2) we could readily expand precursor CTL and demonstrate their cytotoxicity by standard chromium-release assay. We now sought to evaluate the immune reactivity of Ep-2-specific CTL expanded from the peripheral blood mononuclear cells by real-time quantitative polymerase chain reaction assay (LightCycler system) measuring the change in interferon-&ggr; mRNA levels upon stimulation. Cytotoxic T lymphocyte response against the Ep-2 peptide in two patients with epithelial cell adhesion molecule-expressing lung cancer and three healthy donors was compared with the chromium-release assay. In vitro expanded epithelial cell adhesion molecule-specific CTL precursors that exhibited significantly elevated interferon-&ggr; mRNA levels also lysed Ep-2 peptide-pulsed target cells. Our study indicates that quantitative polymerase chain reaction may be a supplement to chromium-release assay for monitoring in vitro expanded CTL precursor reactivity.