Hepatopoietin (HPO) is a novel human hepatotrophic growth factor, which specifically stimulates proliferation of cultured primary hepatocytes in vitro and liver regeneration after liver partial hepatectomy in vivo. Recently, the identification of the mitogenic effect of HPO on hepatoma cell lines and the existence of HPO-specific receptors indicate that HPO acts via its specific cell surface receptor. However, the molecular mechanism of HPO action is not fully elucidated. In this report, we examined the signal transduction events induced by HPO in hepatoma cell line (HepG2). Our results demonstrated that HPO induces phosphorylation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase (MAPK) in a rapid and transient manner. HPO stimulates tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Furthermore, we observed that both MAPK activation and the mitogenic effect of HPO on HepG2 cells were completely blocked by AG1478, a specific inhibitor of EGFR tyrosine kinase activity. However, the effects of HPO were not antagonized by an EGFR-blocking antibody, mAb528, which blocks the interaction between epidermal growth factor and EGFR, indicating that stimulation of tyrosine phosphorylation of EGFR by HPO was not mediated by epidermal growth factor. In contrast, genistein, a general tyrosine kinase inhibitor, significantly attenuated the tyrosine phosphorylation of EGFR in response to HPO. In conclusion, our results suggest that tyrosine phosphorylation of EGFR may play a critical role in MAPK activation and mitogenic stimulation by HPO.