We have studied epidermal growth factor receptor (EGFR) phosphorylation and localization in the pre-replicative phase of liver regeneration induced by a 70% partial hepatectomy (PH), and how a PH affects EGFR activation and trafficking. When Western blotting was performed on livers after PH with antibodies raised against activated forms of EGFR autophosphorylation sites, no marked increase in EGFR tyrosine phosphorylation was observed. However, events associated with attenuation of EGFR signals were observed. Two hours after PH, we found increased EGFR ubiquitination and internalization, followed by receptor downregulation. Furthermore, EGFR phosphorylation following an injection of EGF was reduced after PH. This reduction correlated with an increased activation of PKC and a distinct augmentation in the phosphorylation of the PKC-regulated T654-site of EGFR. When primary cultured hepatocytes were treated with tetradecanoylphorbol acetate (TPA) to induce T654-phosphorylation of EGFR, we found colocalization of a fraction of EGFR with EEA1, downregulation of EGF-mediated EGFR autophosphorylation, altered ligand-induced intracellular sorting of EGFR, and increased mitogenic signaling through the EGFR-Ras-Raf-ERK pathway. Further, we found that both TPA and a PH enhanced EGF-induced proliferation of hepatocytes. In conclusion, our results suggest that hepatocyte priming involves modulation of EGFR that enhances its ability to mediate growth factor responses without an increase in its receptor tyrosine kinase-activity. This may be a pre-replicative competence event that increases growth factor effects during G1 progression.