In cultured rat hepatocytes, the gluconeogenic key enzyme, phosphoenolpyruvate carboxykinase (PCK), is induced by glucagon via elevation of cyclic 3′,5′ adenosine monophosphate (cAMP). The proinflammatory cytokine, interleukin-6 (IL-6), which in the liver together with IL-1β and tumor necrosis factor α triggers the acute-phase response, had been shown to attenuate the glucagon-induced increase in PCK gene transcription, messenger (mRNA) levels, and enzyme activity. The molecular mechanism of this inhibition was investigated in the present study. Glucagon increased cyclic cAMP and PCK mRNA levels to a transient maximum twofold and fivefold, respectively. The increases were attenuated by IL-6. Forskolin, which stimulates adenylate cyclase activity, increased cAMP and PCK mRNA levels 1.6-fold and fivefold, respectively. However, IL-6 attenuated the forskolin- stimulated increase in PCK mRNA but not the increase in cAMP. This showed that IL-6 inhibited PCK mRNA increase in part by the attenuation of cAMP increase, but also beyond cAMP formation. This was confirmed in experiments in which PCK mRNA levels were increased by the nonhydrolyzable cAMP-analogue, chlorophenylthio (CPT)-cAMP. The increase in PCK mRNA was again attenuated by IL-6. In pertussis toxin- and in isobutylmethylxanthine-treated hepatocytes, IL-6 still inhibited the glucagon-stimulated increase in cAMP, indicating that IL-6 did not activate an inhibitory G-protein or phosphodiesterase, which could cause the impairment of cAMP increase. To demonstrate whether the inhibition of PCK gene expression by IL-6 beyond cAMP might be caused by the inhibition of the activation of the PCK gene promoter by cAMP, cultured rat hepatocytes were transfected with a luciferase reporter gene construct under the control of a PCK gene promoter fragment (base −979 to base +32). Luciferase activity was determined after stimulation of the cells with CPT-cAMP in the absence or presence of IL-6. CPT-cAMP increased luciferase activity by 1.7-fold, which was inhibited in the presence of IL-6. It is concluded that IL-6 had a dual inhibitory effect on the stimulation of PCK gene expression by glucagon. It inhibited the increase in cAMP at a site before cAMP formation by adenylate cyclase and at a site after cAMP formation, the activation of the PCK gene promoter by cAMP.