Increased intracellular cyclic adenosine monophosphate (cAMP) levels have been shown in some reports to inhibit and in other studies to stimulate growth factor-mediated activation of the mitogen-activated protein kinase (MAP kinase) pathway, depending on the cell type examined. The relationship between cAMP and MAP kinase in hepatocytes has not been examined. In the current study, stimulation of primary cultures of rat hepatocytes with hepatocyte growth factor (HGF) or epidermal growth factor (EGF) increased Ras, Raf, and MAP kinase activity. Incubation of hepatocytes with cAMP-increasing agents blocked activation of Raf by both HGF and EGF, whereas activation of Ras was unaffected. MAP kinase activation by HGF was completely inhibited, whereas EGF-stimulated MAP kinase activity was only slightly reduced. Incubation of hepatocytes with pertussis toxin slightly blunted MAP kinase activation by EGF but not HGF. Increasing cAMP in hepatocytes preincubated with pertussis toxin completely inhibited the activation of MAP kinase by EGF. In conclusion, HGF activates MAP kinase in hepatocytes exclusively through an Raf-dependent pathway and this activation may be completely blocked by increasing cAMP. In contrast, EGF activates MAP kinase in hepatocytes through both Raf-dependent and Raf-independent pathways: the latter pathway probably involves a pertussis toxin-sensitive G protein.