Many hormones regulate the rate of synthesis of phosphoenolpyruvate carboxykinase (PEPCK), the enzyme that governs the rate-limiting step in gluconeogenesis. In H4IIE rat hepatoma cells, glucocorticoids, retinoic acid and cyclic AMP (cAMP) increase PEPCK gene transcription whereas insulin and phorbol esters have the opposite effect. Insulin and phorbol esters are dominant as they prevent cAMP- and glucocorticoid-stimulated PEPCK gene transcription. In contrast, insulin and phorbol esters both stimulate transcription of gene 33 in the same H4IIE cells, with the same time course as seen for their inhibitory effect on PEPCK gene transcription. We now report that the protein phosphatase inhibitor, okadaic acid, mimics the action of insulin and phorbol esters on expression of both gene 33 and PEPCK gene in H4IIE cells. Okadaic acid stimulates gene 33 mRNA accumulation whereas it inhibits cAMP- and glucocorticoid-stimulated PEPCK mRNA accumulation. The effect of okadaic acid on the PEPCK gene is mediated through the PEPCK promoter as, in a cell line, HL1C, stably transfected with a PEPCK-chloramphenicol acetyltransferase (CAT) fusion gene, okadaic acid inhibits cAMP- and glucocorticoid-stimulated CAT expression. Desensitization of the protein kinase C pathway by exposure to phorbol 12-myristate 13-acetate for 16 h abolishes the subsequent action of the phorbol ester but does not markedly affect the inhibition of cAMP- and glucocorticoid-stimulated CAT expression by insulin or okadaic acid. Even though insulin and okadaic acid appear to repress PEPCK gene expression through a pathway initially distinct from that used by phorbol esters, transient-transfection studies show that the final target of the action of okadaic acid, insulin and phorbol ester is the same DNA element.