In the present study, we investigated the role of calcium and protein kinase C (PKC) in the activation of mitogen-activated protein kinase (MAPK) in isolated rat hepatocytes. We found that the glycogenolytic hormone norepinephrine (NE), acting through the alpha1-adrenergic receptor and the G protein Gq, was able to induce a dose- and time-dependent activation of MAPK in hepatocytes. Vasopressin, which acts through a different receptor but also through stimulation of the Gq-dependent pathway, also caused a twofold activation of MAPK. Activation of MAPK by both agonists required the presence of free extracellular calcium and was blocked by the specific PKC inhibitor, Ro 31-8220. MAPK activation was also induced by phorbol myristate acetate (PMA), confirming that a PKC-dependent pathway exists for MAPK activation in liver. Furthermore, calcium-mobilizing agents such as thapsigargin and ionomycin were able to induce an activation of MAPK by a PKC-independent pathway that was totally abolished by preincubation of cells with EGTA. A second pathway for MAPK activation that relies solely on calcium may therefore exist. Ro 31-8220 did not affect phosphorylase activation by NE, vasopressin, thapsigargin, and ionomycin, indicating that PKC inhibition did not interfere with the signaling pathway leading to inositol-1,4,5-triphosphate (IP3)-induced calcium mobilization or with changes in calcium fluxes. The role of MAPK activation by NE and vasopressin in the regulation of hepatic carbohydrate metabolism is discussed.