In the current study, increased interferon (IFN)-γ, interleukin (IL)-10, and IL-12 p40 serum levels were observed in patients with chronic hepatitis C (CHC) compared to controls. Patients also displayed an increased spontaneous IFN-γ release but a deficient peripheral blood mononuclear cells (PBMC) IFN-γ production following stimulation with Staphylococcus aureus Cowan I strain (SAC). No difference was found with reference to spontaneous or phytohaemagglutinin (PHA)-induced IL-10 release between patients and controls, whereas a higher IL-12 p70 and IL-12 p40 secretion triggered by SAC was observed in patients. Moreover, IL-12 p40/p70 ratio following SAC stimulation was higher in patients compared to controls and a negative correlation was found between this ratio and IFN-γ amounts. Recombinant IL-12 (rIL-12) as well as neutralizing anti-IL-10 monoclonal antibodies (mAbs) were able to restore the compromised IFN-γ production. Of note, anti-IL-10 supplementation induced a lower IL-12 p40/p70 ratio in HCV subjects as compared to controls. Finally, IFN-α upregulated in vitro IFN-γ, IL-10, and IL-12 p70 release but not IL-12 p40 secretion, this giving rise to a normalization of IL-12 p40/p70 ratio. The data suggest the occurrence of an enhanced responsiveness to IL-10 modulating effects, likely mediated by an imbalance of IL-12 p40/p70 ratio, in chronic HCV infection. Cytokine balance restoration might thus contribute to achieve therapeutical results in chronic hepatitis C.