Immunosuppression considerably affects hepatitis C virus (HCV) recurrence and the outcome of antiviral treatment after liver transplantation. Recent findings have suggested that the calcineurin inhibitor tacrolimus (Tac), unlike cyclosporine A (CsA), interferes with the antiviral activity of interferon-alpha (IFN-alpha) in vitro. The aim of this study was to more extensively investigate the effects of calcineurin inhibitors on IFN-alpha signaling and antiviral activity in subgenomic and infectious HCV models. Treatment with Tac and CsA did not affect Huh7 cell proliferation at doses of 10 to 500 ng/mL; however, it completely inhibited T cell proliferation. In contrast to previous reports, Tac had no effect on IFN-alpha-stimulated reporter gene expression, even at the dose of 5 mu g/mL. Furthermore, in Huh7 subgenomic HCV replicon cells, treatment with Tac had no significant effect on the suppression of viral replication by IFN-alpha. In the infectious HCV model, treatment with IFN-alpha effectively inhibited both viral RNA replication and de novo production of virus particles, and neither was attenuated at any concentration of Tac. CsA had no significant effect on IFN-alpha-stimulated reporter gene expression; however, as shown previously, a combination of CsA (at 500 ng/mL and higher) and IFN-a resulted in enhanced inhibition of viral replication in both the subgenomic and infectious HCV models. In conclusion, our study shows no evidence that Tac or CsA interferes with IFN-alpha-mediated inhibition of HCV replication and virion production in vitro. Therefore, no further mechanistic arguments have been found to break the clinical controversy about the choice of calcineurin inhibitors during posttransplantation antiviral therapy. Liver Transpl 16:520-526, 2010. (C) 2010 AASLD.