The multihormonal regulation of phosphoenolpyruvate carboxykinase (PEPCK) was studied using chimeric genes composed of various regions of the PEPCK gene promoter region fused to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene. These constructions, transfected into H4IIE hepatoma cells, are regulated like the endogenous PEPCK gene: dexamethasone and cAMP both stimulate PEPCK-CAT gene expression and their effects are additive; insulin inhibits the individual or combined effects of these stimulatory agents; and insulin inhibits dexamethasone-stimulated PEPCK-CAT fusion gene expression in a concentration-dependent fashion that is half-maximal at 10(-11) M. The induction by dexamethasone and the inhibition by insulin is specific for the DNA sequences that flank the 5' end of the PEPCK gene because similar effects were not observed for a plasmid in which the promoter and enhancer sequences of simian virus 40 (SV40) are fused to CAT. These results imply that the DNA adjacent to the transcription start site of the PEPCK gene contains the cis-acting hormone response elements responsible for the multihormonal regulation of this gene, including the insulin response.