The mechanism(s) by which interferon (IFN)-alpha confers resistance to viruses is currently being characterized. Previous studies have shown that binding of IFN-alpha to its high-affinity receptor activates transcription factor interferon-stimulated gene factor 3 (ISGF3), which positively regulates a number of antiviral genes including 2'-5'-oligoadenylate synthetase (2-5A synthetase). We show that mouse L cells expressing nonsecreted (intracellular) type I human IFN are less susceptible to encephalomyocarditis (EMC) virus infection and have increased levels of 2-5A synthetase. The 2-5A synthetase promoter is constitutively induced, and the antiviral effects are most likely mediated through activation of ISGF3, which occurs constitutively in cell lines expressing intracellular interferon. These data suggest that the internalization of IFN-alpha may play a role in the antiviral properties associated with IFN.