Alcohol consumption accelerates liver damage and diminishes the anti-hepatitis C virus (HCV) effect of interferon alfa (IFN-α) in patients with HCV infection. It is unknown, however, whether alcohol enhances HCV replication and promotes HCV disease progression. The availability of the HCV replicon containing hepatic cells has provided a unique opportunity to investigate the interaction between alcohol and HCV replicon expression. We determined whether alcohol enhances HCV RNA expression in the replicon containing hepatic cells. Alcohol, in a concentration-dependent fashion, significantly increased HCV replicon expression. Alcohol also compromised the anti-HCV effect of IFN-α. Investigation of the mechanism(s) responsible for the alcohol action on HCV replicon indicated that alcohol activated nuclear factor κB (NF-κB) promoter. Caffeic acid phenethyl ester (CAPE), a specific inhibitor of the activation of NF-κB, abolished alcohol-induced HCV RNA expression. In addition, naltrexone, an opiate receptor antagonist, abrogated the enhancing effect of alcohol on HCV replicon expression. In conclusion, alcohol, probably through the activation of NF-κB and the endogenous opioid system, enhances HCV replicon expression and compromises the anti-HCV effect of IFN-α. Thus, alcohol may play an important role in vivo as a cofactor in HCV disease progression and compromise IFN-α-based therapy against HCV infection.