Although hepatitis C virus (HCV) has been shown to readily escape from virus-specific T and B cell responses, its effects on natural killer (NK) cells are less clear. Based on two previous reports that recombinant, truncated HCV E2 protein inhibits NK cell functions via crosslinking of CD81, it is now widely believed that HCV impairs NK cells as a means to establish persistence. However, the relevance of these findings has not been verified with HCV E2 expressed as part of intact virions. Here we employed a new cell culture system generating infectious HCV particles with genotype 1a and 2a structural proteins, and analyzed direct and indirect effects of HCV on human NK cells. Antibody-mediated crosslinking of CD16 stimulated and antibody-mediated crosslinking of CD81 inhibited NK cell activation and interferon gamma (IFN-γ) production. However, infectious HCV itself had no effect even at titers that far exceeded HCV RNA and protein concentrations in the blood of infected patients. Consistent with these results, anti-CD81 but not HCV inhibited NK cell cytotoxicity. These results were independent of the presence or absence of HCV-binding antibodies and independent of the presence or absence of other peripheral blood mononuclear cell populations. Conclusion: HCV 1a or 2a envelope proteins do not modulate NK cell function when expressed as a part of infectious HCV particles. Without direct inhibition by HCV, NK cells may become activated by cytokines in acute HCV infection and contribute to infection outcome and disease pathogenesis.