We have studied the antiviral activities of five recombinant interferon-alpha (IFN-alpha) subtypes, namely IFN-alpha1, -alpha2, -alpha5, -alpha8 and -alpha10, in eight human liver cancer cell lines. The relative antiviral activities, expressed in terms of the mean 50% inhibitory concentration (IC50), were different for each cell line. In general, IFN-alpha8 was the most potent, IFN-alpha2, -alpha5, and -alpha10 were intermediately active, and IFN-alpha1 was the least potent in the all cell lines. The observed differences between the IC50s of IFN-alpha1 and -alpha8 ranged from 250- to 2200-fold in these cell lines. Thus, the ranking order of relative antiviral activity was similar but the sensitivity to the subtypes was different among these cell lines. The relative antiviral activities of the subtypes were associated with the induction of 2',5'-oligoadenylate synthetase (2',5'-OAS) in the typical hepatocellular carcinoma cell line HAK-3 but not in the cell line KYN-3. Next, we examined for synergistic antiviral activity induced by IFN-alpha2 and -alpha8 that has been reported for the hepatocellular carcinoma cell line, HepG2. Synergism was observed in three of the eight liver cell lines at an IFN-alpha2 to -alpha8 ratio of 60:40, and is considered to reflect the synergistic induction of 2',5'-OAS.