Previously, we reported that serotonin (5-hydoxytryptamine; 5-HT) induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. The 5-HT effect was due to transforming growth factor (TGF)-α secreted by activation of 5-HT_2B receptor/phospholipase C (PLC)/Ca²⁺ pathway. In this study, we investigated whether 5-HT would stimulate phosphorylation of ribosomal p70 S6 kinase (p70S6K) in the cultured cells. Phosphorylated p70S6K was identified by Western blotting analysis using anti-phospho-p70S6K monoclonal antibody. The phosphorylated p70S6K was increased at 5 min, and reached a peak at 30 min after 5-HT addition. On the other hand, the phosphorylation of p70S6K induced by 5-HT was completely abolished the 5-HT_2B receptor antagonist, LY272015, U-73122, a PLC inhibitor, BAPTA/AM, a membrane-permeable Ca²⁺ chelator, verapamil, L-type Ca²⁺ channel blocker, and somatostatin. Moreover, specific inhibitors of growth-related signal transducers (e.g., LY294002, PD98059, and rapamycin) blocked phosphorylation of p70S6K induced by 5-HT. These results suggest that secretion of TGF-α accelerates hepatocyte proliferation through the mitogen-activated protein kinase (MAPK)/p70S6K pathway.