Summary. Chronic hepatitis C virus (HCV) infection is characterized by attenuated antiviral T-cell responses, making their detection and characterization a technological challenge. The role and the dynamics of antiviral T-cell responses during antiviral therapy are incompletely understood. To assess HCV-specific T-cell responses during antiviral therapy of genotype-1-infected patients, we adopted a flow cytometric approach to comprehensively evaluate virus-specific CD4+ and CD8+ T-cell proliferative responses against pools of genotype- and subtype-specific serial, overlapping peptides spanning the entire virus. Studies in cross-sectional cohorts of treatment-naïve (TN) patients , early and sustained clinical virological responders (EVRs and SVRs) or clinical nonresponders (NRs) showed that this proliferative assay had significantly greater sensitivity in detecting HCV-specific responses, compared with ex vivo cytokine flow cytometry. At the same time, it could be used to detect and quantify both CD4+ and CD8+ responses simultaneously. EVRs and SVRs showed significantly more HCV-specific CD4+ and CD8+ responses, compared with either TN patients or NRs. This corresponded to a higher magnitude of responses as well as a greater breadth of reactivity with higher responses against the core/E1, NS3, NS4 and NS5b regions of the virus. Interestingly, both clinical responders and NRs showed higher cytomegalovirus-specific CD4+ responses, compared with TN patients. These results demonstrate an association between clinically successful antiviral therapy and enhanced magnitude and breadth of antiviral responses. Moreover, the study demonstrates the clinical relevance of this flow cytometric proliferation assay system, in combination with an unbiased library of viral peptides, in evaluating the biology of antiviral T-cell responses during infection and therapy.