Intrahepatic virus-specific CD8(+) T cells are thought to be important for the control of hepatitis C virus (HCV) infection, yet the precise kinetics for the expansion of epitope-specific T cells over the course of infection are difficult to determine with currently available methods. We used a real-time PCR assay to measure the frequency of clonotypic HCV-specific CD8(+) T cells in peripheral blood and snap-frozen liver biopsy specimens of two chimpanzees (Pan troglodytes) with previously resolved HCV infection who were rechallenged with HCV. In response to rechallenge, the magnitude of each clonotypic response was 10-fold higher in the liver than in the blood, and the peak clonotype frequency was concurrent with the peak viral load. The higher frequency of HCV-specific clonotypes in the liver than in peripheral blood was maintained for at least 3 months after the clearance of viremia. After antibody-mediated CD8(+) T-cell depletion and another viral challenge, the rebound of these clonotypes was seen prior to an appreciable reconstitution of CD8(+) T-cell values and, again, at higher frequencies in the liver than in peripheral blood. These data demonstrate the importance of intrahepatic virus-specific CD8(+) T cells for the clearance of infection and the rapid kinetics of expansion after virus challenge.