Three human hepatoma cell lines, PLC/PRF/5, Mahlavu and Sk-Hep 1, two of which contain integrated HBV DNA, were grown in culture and treated with human alpha-IFN for up to 14 days. IFN treatment caused a varying suppression of cell growth of the three hepatoma cell lines. While doubling time and cloning efficiency were significantly reduced for all three hepatoma cell lines tested, 3[H]thymidine incorporation was markedly suppressed, in a dose-dependent fashion, only in treated PLC/PRF/5 cells but not in Sk-Hep 1 and Mahlavu cells. The inhibiting effect of interferon treatment on growth of PLC/PRF/5 cells in vitro was neutralized by antibodies to human IFN. IFN treatment caused a significant suppression of HBsAg and alpha FP secretion by PLC/PRF/5 hepatoma cells. This effect, while constant throughout the observation period for HBsAg, was cumulative for alpha FP secretion. Following discontinuation of treatment, suppression of PLC/PRF/5 hepatoma cell growth was rapidly reversed, and HBsAg and alpha FP secretion returned to their pretreatment levels. These experiments suggest that human alpha-IFN suppresses the growth of some human hepatoma cells in culture but that this effect is dependent on the continuous presence of IFN in the growth medium. Finally, the inhibitory effects of IFN on cell growth differed for the various hepatoma cell lines tested.