It is believed that the hepatitis C virus (HCV)-specific CD8⁺ cytotoxic T lymphocytes (CTLs) play a role in the development of liver cell injury and in the clearance of the virus. To develop a direct binding assay for HCV-specific CTLs, we generated two peptide-MHC tetramers by using the recombinant HLA A2.1 molecule and A2-restricted T cell epitopes of the HCV NS3 protein. With these reagents we are able to detect specific CD8⁺ cells in the blood of 15 of 20 HLA-A2⁺, HCV-infected patients, at a frequency ranging from 0.01% to 1.2% of peripheral CD8⁺ T cells. Phenotypic analysis of these specific cells indicated that there is a significant variation in the expression of the CD45 isoforms and CD27 in different patients. A 6-hour incubation of one patient’s blood with NS3 peptides resulted in the activation of the epitope-specific CD8⁺ cells, as indicated by their expression of CD69 and IFN-γ. We also detected NS3-specific CD8⁺ T cells in the intrahepatic lymphocyte population isolated from liver biopsies of two HCV-infected patients. The frequency of these specific CD8⁺ cells in the liver was 1–2%, at least 30-fold higher than in the peripheral blood. All of the intrahepatic NS3-specific CD8⁺ T cells were CD69⁺, suggesting that they were activated CTLs. Direct quantitation and characterization of HCV-specific CTLs should extend our understanding of the immunopathogenesis and the mechanism of clearance or persistence of HCV.