We investigated the effects of pegylated (PEG)-IFN-alpha2b on alpha-fetoprotein (AFP) expression as demonstrated by protein and mRNA levels in six human hepatocellular carcinoma (HCC) cell lines. The number of KIM-1 cells in culture with PEG-IFN-alpha2b decreased between 24 amd 240 h, whereas the levels of intracellular and secreted AFP per cellular protein increased (except at 192 h), with levels 1.9-fold and 2.9-fold higher at maximum, respectively, than cells without PEG-IFN-alpha2b (control). The mRNA level increased between 72 and 192 h, when the level was 3-fold higher than that of the control. In the 72-h culture with 40-5000 IU/mL PEG-IFN-alpha2b, there were dose-dependent increases in AFP protein and mRNA expression and dose-dependent decrease in cell number resulting from apoptosis and blockage of the cell cycle at the S-phase. The rate of fucosylated AFP in the cell lysate decreased in a dose-dependent and time-dependent manner. In the PEG-IFN-alpha2b culture of the other five HCC cell lines, cell proliferation was suppressed, but the expressions of AFP protein and mRNA increased in only two cell lines, and suppression of cell proliferation was not related to the increase in AFP expressions. Our findings demonstrated that PEG-IFN-alpha2b induces an increase in AFP expression at both the protein and mRNA levels.