The participation of phosphatidylinositol 3–kinase (PI3-kinase), protein kinase C, and mitogen-activated protein kinase (MAP-kinase) in the inhibition by interleukin 6 (IL-6) and insulin of phosphoenolpyruvate carboxykinase (PCK) gene expression was investigated in cultured rat hepatocytes. IL-6 or insulin inhibited the glucagon-stimulated increase in PCK messenger RNA (mRNA) by about 70%. In the presence of either the PI3-kinase inhibitor, wortmannin, or the protein kinase C inhibitor, GF109203x, the inhibition by IL-6 was only about 40%, although it was abolished with both inhibitors in combination. Wortmannin alone but not GF109203x prevented the inhibition by insulin of glucagon-stimulated PCK gene expression. The MAP-kinase pathway inhibitor, PD98059, did not affect IL-6 or insulin inhibition of PCK mRNA increase. When chlorophenylthio–cyclic 3′,5′ adenosine monophosphate (CPT-cAMP) was used instead of glucagon, IL-6 or insulin inhibited the increase in PCK mRNA by 75% and 85%, respectively. The inhibition by IL-6 was only about 50% in the presence of either wortmannin or GF109203x alone but was abolished with the combination of both inhibitors. The inhibition by insulin was only about 50% in the presence of GF109203x and was abolished by wortmannin. The inhibitors did not affect the inhibition by IL-6 or insulin of the glucagon-stimulated increase in cAMP. It is concluded that the inhibition by IL-6 of PCK gene expression involved both PI3-kinase and protein kinase C, whereas the inhibition by insulin required only PI3-kinase. The inhibition occurred downstream from cAMP formation. Hence, IL-6 and insulin may share, in part, common signal transduction pathways in the inhibition of PCK gene expression.