Epidermal growth factor (EGF) stimulates freshly plated adult hepatocytes to synthesize DNA, but only after they pass through a lag phase of 40 hours following EGF exposure. The longer the cells are maintained, they become more responsive to EGF and the lag phase shortens. Maximal EGF-mediated stimulation of DNA synthesis requires the induction of ErbB2, which is not normally expressed in adult hepatocytes. We used immunologic methods to demonstrate increased expression during culture of two gene families required for EGF to stimulate hepatocyte DNA synthesis: Akt and ERK 1,2. Both gene families showed hyperexpression in culture particularly when cells were exposed to insulin and EGF. Unlike CDK-2 and cyclin D1, integral mediators of the G1/S phase transition, ERK 1,2 and AKT appeared in the absence of EGF, particularly when insulin was present. This hyperexpression, which high concentrations of dexamethasone reversed, increased basal and growth factor-stimulated phosphorylation of Akt and ERK 1,2. Pharmacologic blockade of phosphatidylinositol kinase (PI-3K) suppressed the Akt increase whereas pharmacologic blockade or siRNA downregulation of ErbB2 inhibited both Akt and ERK 1,2 synthesis. All three Akt isoforms contributed to the increase in total Akt. EGF but not insulin specifically upregulated Akt 2 and 3. Since Akt and ERK 1,2 are also hyperexpressed in poorly differentiated hepatomas, their dysregulation in cancer may involve transcriptional mechanisms normally operative in cultured hepatocytes. We hypothesize that the induction and activation of ErbB2 increases the expression of these kinases, enhancing the responsiveness of hepatocytes to EGF as they adapt to culture.