Chronic hepatitis C is characterized by a weak or absent hepatitis C virus (HCV)-specific CD4+ T-cell response in terms of antigen-specific proliferation or interferon gamma (IFN-γ) secretion. To clarify whether this is due to the absence or functional impairment of antigen-specific CD4+ T cells we developed an assay that relies on the induced expression of the T-cell activation marker CD25 and is therefore independent from cytokine secretion or proliferation. In 10 of 20 patients with chronic hepatitis C, a significant number of antigen-specific activated CD4+ T cells (mean 1.06%/patient; range, 0% to 5.2% of CD4+ T cells) could be shown, whereas antigen-specific proliferation was present in only 1 of 20 patients. IFN-γ secretion was absent in all 13 patients tested. However, significant antigen-specific interleukin 10 (IL-10) and transforming growth factor β (TGF-β) secretion was present in 6 of 10 and 3 of 10 patients, respectively. In 8 patients with acute hepatitis C, irrespective of disease outcome, HCV-specific CD4+ T cells were detected in all patients and at a significantly higher frequency (mean 3.7%/patient; range, 1.16% to 7.17%) in the first weeks of disease. A chronic course of disease was associated either with a loss of both IFN-γ secretion and proliferation, resembling an anergic state, or a loss of T-cell proliferation followed by a rapid decline in IFN-γ-producing cells, corresponding to exhaustion of the specific immune response. In conclusion, functional changes of HCV-specific CD4+ T cells or failure to develop a long-lasting T-helper response may contribute to chronic hepatitis C viral persistence.