ABSTRACT: The detection of hepatitis C virus (HCV)-specific T cell responses in HCV-uninfected, presumably unexposed, subjects could be due to an underestimation of the frequency of spontaneously resolving infections, as most acute HCV infections are clinically silent. To address this hypothesis, HCV-specific cellular immune responses were characterized, in individuals negative for an HCV PCR assay and humoral response, with (n=32) or without (n=33) risk of exposure to HCV. Uninfected volunteers (n=20) with a chronically HCV-infected partner were included as positive controls for potential exposure to HCV and HCV infection, respectively. HCV-specific T cell responses in freshly isolated peripheral blood mononuclear cells were studied ex vivo by ELISPOT and CFSE-based proliferation assays using panels of HCV Core and NS3-derived peptides. A pool of unrelated peptides was used as a negative control, and a peptide mix of human cytomegalovirus, Epstein-Bar virus and Influenza virus as a positive control. Overall, 20% of presumably HCV-uninfected subject tested had detectable T-cell responses to the virus, a rate much higher than previous estimates of HCV prevalence in developed countries. This result would be consistent with unapparent primary HCV infections that either cleared spontaneously or remained undetected by conventional serological assays.