While interferon-alpha (IFN-alpha) subtypes share a common specific receptor composed of two subunits, interferon-alpha receptor (IFNAR)-1 and IFNAR-2, their subtype activities are exhibited via several intracellular signaling pathways and thus subsequently show different biological effects. Anti-proliferative effects of single treatment with IFN-alpha subtypes or 5-fluorouracil (FU), and of combined treatment with each IFN-alpha subtype and 5-FU were examined on three hepatocellular carcinoma cell lines, HepG2, HLE and PLC/PRF/5. HepG2 and PLC/PRF/5 cells were susceptible to the combination treatment, but HLE cells were not. Proliferation of PLC/PRF/5 cells was also inhibited by the IFN-alpha subtypes singly. In addition, apoptosis was observed in HepG2 cells upon treatment with 5-FU alone and with the combination treatment, and in PLC/PRF/5 cells after single treatment with the IFN-alpha subtypes and after the combination treatment. IFN-alpha subtypes induced cell cycle arrest in the G2/M phase in HepG2 and PLC/PRF/5. Analyses by Western blotting and immunoprecipitation revealed increased p53 phosphorylation in HepG2 and PLC/PRF/5 cells but not in HLE cells after combined treatment. Single treatment with IFN-alpha subtypes promoted p53 activation only in PLC/PRF/5 cells. These results propose that IFN-alpha subtypes induce cells to undergo apoptosis through p53 activation directly and indirectly, in collaboration with 5-FU, further suggesting the presence of distinct signal pathways for IFN-alpha-induced apoptosis.