Cytotoxic T lymphocytes have been reported to be involved in the immune clearance of virus-infected cells and in the pathogenesis of viral infection. W e studied the cytotoxic T lymphocyte response to the putative nucleocapsid protein of hepatitis C virus in patients with chronic hepatitis C. Cytotoxic T lymphocytes specific for hepatitis C virus nucleocapsid protein were generated from peripheral blood lymphocytes by means of repeated stimulation with a synthetic hepatitis C virus nucleocapsid protein peptide. The cytotoxic T lymphocytes were CD8 positive and recognized an epitope in hepatitis C virus nucleocapsid protein residues 81 to 100 in association with a human leukocyte antigen class I molecule, B44. The peptideinduced cytotoxic T lymphocytes recognized target cells synthesizing hepatitis C virus nucleocapsid protein endogenously, though less efficiently than peptide-pulsed target cells. The human leukocyte antigen B44-restricted cytotoxic T lymphocyte response was observed in three of five patients with chronic hepatitis C and a human leukocyte antigen B44 molecule but in neither of two hepatitis C virus-negative healthy individuals with human leukocyte antigen B44 molecules. The results demonstrate the presence of hepatitis C virus-specific cytotoxic T lymphocytes in the peripheral blood of patients with chronic hepatitis C and provide a strategy to study the role of cytotoxic T lymphocytes in the viral clearance and the pathogenesis of hepatitis C virus infection. (HEPATOLOGY 1993;18: 1039-1044.)