Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8⁺ T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4⁺CD25⁺ regulatory phenotype in suppressing virus-specific CD8⁺ T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8⁺ T cells were inhibited by CD4⁺CD25⁺ T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8⁺ T cells but also to influenza virus-specific CD8⁺ T cells. Importantly, CD4⁺CD25⁺ T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8⁺ T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4⁺CD25⁺ cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4⁺CD25⁺ T cells that are able to suppress CD8⁺ T-cell responses to different viral antigens. Our results further suggest that CD4⁺CD25⁺ T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.