Ribavirin is an important component of interferon-based and direct antiviral treatment regimens for hepatitis C virus (HCV) infection. Immunomodulation, in particular improvement of the host interferon (IFN) response, has been proposed as ribavirin’s mechanism of action. Natural killer (NK) cells are sensitive biomarkers for IFN-α/β receptor signaling, as NK cell cytotoxicity and IFN-γ production are regulated by STAT1- and STAT4-phosphorylation, respectively. Specifically, pSTAT1-dependent NK cell cytotoxicity increases and pSTAT4-dependent IFN-γ production decreases in response to endogenous, virus-induced IFN-α and during IFN-α-based therapy. To assess whether ribavirin has a direct effect on NK cells and/or improves the IFN-γ response of NK cells in the presence of IFN-α, we prospectively studied 22 HCV patients with and 32 patients without 4 weeks of ribavirin pretreatment, who all received subsequent PegIFN/ribavirin combination therapy. During ribavirin pretreatment, the frequency of CD56dim NK cells with cytotoxic effector functions decreased (p=0.049) as did the frequency of CD56bright NK cells with the capacity to produce IFN-γ (p=0.001). In vitro or in vivo exposure of NK cells to ribavirin improved the pSTAT4 (p