Although aflatoxin B(1) (AFB(1)) is known as a mycotoxin that induces hepatocellular carcinoma (HCC), its effects on HCC cells have not been sufficiently investigated. The HCC cell lines HepG2, Huh-6, Huh-7, and PLC were cultured (5 x 10(5)cells/ml) and various concentrations of AFB(1) were added. The expression levels of the alpha-fetoprotein (AFP), insulin-like growth factor-2 (IGF-2), and insulin-like growth factor-1 receptor (IGF-1R) genes in each sample were determined by real-time PCR, with the following results: (1) The level of AFP expression in HepG2 increased at 5-50 ng/ml of AFB(1) in a dose-dependent manner. The AFP expression level in Huh-6 increased at 0.01-5 ng/ml of AFB(1) in a dose-dependent manner and decreased to half controls level at 50 ng/ml of AFB(1). The AFP expression level in Huh-7 decreased to one-third the original level at 0.5-50 ng/ml of AFB(1). The AFP expression level in PLC decreased at 0-0.5 ng/ml of AFB(1) in a dose-dependent manner, and decreased to one-third at concentrations of AFB(1) between 0.5 and 50 ng/ml. (2) The IGF-2 and IGF-1R expression levels in Huh-6 increased more than 10-fold at 0.5-5 ng/ml of AFB(1), but decreased to half at 50 ng/ml of AFB(1). The IGF-2 and IGF-1R expression levels in other cell lines increased in a dose-dependent manner. AFB(1) induced translations of IGF-2 and IGF-1R and cell proliferation: When 50 ng/ml AFB(1) was administrated, cell numbers were 2.0-, 1.7-, and 1.5-fold higher than those of controls after 3 days of culture in HepG2, Huh-7, and PLC, respectively. Particularly, in Huh-6, it increased 2.5-fold higher than those of controls following 5 ng/ml AFB(1) administration. The ratio of fold-change phospho-IGF-1R in all cell lines that were treated with AFB(1), increased 1.1-1.5-fold. These results indicate that AFB(1) may enhance HCC cell proliferation through an IGF-2-dependent signal axis, although it remains to be investigated whether those effects are associated with human hepatocarcinogenesis resulting from AFB(1) exposure.