CD4⁺CD25⁺ T regulatory cells may play a role in the different clinical presentations of chronic hepatitis C virus (HCV) infection by suppressing CD4⁺ T cell responses. Peripheral CD4⁺CD25⁺ T cells from chronic HCV carriers with normal and abnormal alanine aminotransferase (ALT) were analysed for specificity and effect on HCV-specific CD4⁺ T cell reactivity by flow cytometry for intracellular cytokine production and proliferation assay. HCV-specific CD4⁺CD25^+high T cells consistently produced transforming growth factor (TGF)-β but only limited amounts of interleukin (IL)-10 and no IL-2 and interferon (IFN)-γ. The HCV-specific TGF-β response by CD4⁺CD25^+high T cells was significantly greater in patients with normal ALT compared to patients with elevated ALT. In addition, a significant inverse correlation was found between the HCV-specific TGF-β response by CD4⁺CD25^+high T cells and liver inflammation. In peripheral blood mononuclear cells (PBMC), both HCV antigen-induced IFN-γ production and proliferation of CD4⁺ T cells were greater in patients with elevated ALT compared with patients with normal ALT. Depletion of CD4⁺CD25⁺ cells from PBMC resulted in an increase of both IFN-γ production and proliferation of HCV-specific CD4⁺ T cells that was significantly greater in patients with normal ALT levels compared with patients with elevated ALT. In addition, CD4⁺CD25⁺ T cells from patients with normal ALT levels proved to be significantly more potent to suppress CD4⁺ T cell reactivity with respect to those from patients with elevated ALT. In conclusion, these data support the hypothesis that CD4⁺CD25⁺ cells may play a role in controlling chronic inflammatory response and hepatic damage in chronic HCV carriers.