Hepatitis C virus (HCV) infections were evaluated in chimpanzees that had previously cleared HCV and were rechallenged. Animals that had previously cleared HCV infection rapidly cleared homologous and heterologous virus upon rechallenge, indicative of a strong protective immunity. In one animal, sterilizing immunity was observed with regard to viremia, although viral RNA was transiently detected in the liver. Accelerated viral clearance following rechallenge with HCV was observed in animals that had not been exposed to HCV for over 16 years, suggesting that long-lasting protective immunity may be possible. The ability of peripheral blood mononuclear cells (PBMC) to recognize HCV proteins was evaluated during the course of the rechallenge experiments. A very early and strong in vitro recall response to HCV nonstructural proteins appeared to be associated with viral clearance. In contrast, proliferative responses to HCV proteins were not observed in 4 persistently infected chimpanzees, and a weak proliferative response was observed in 1 of 2 animals during acute resolving infection. The results suggest that a strong T-cell proliferative response is induced upon rechallenge of chimpanzees with HCV and that this response is associated with rapid viral clearance. The antibody response to HCV proteins increased by over 1,000-fold in all animals following rechallenge as well. A more complete understanding of the role of the cellular immune response in the clearance of HCV and the nature of the protective immune response following viral clearance may aid in the generation of therapies and vaccines.