Phosphoenolpyruvate carboxykinase (PEPCK) governs the rate-limiting step in gluconeogenesis. Glucocorticoids and cAMP increase PEPCK gene transcription and gluconeogenesis, whereas insulin and phorbol esters have the opposite effect. Insulin and phorbol esters are dominant, since they prevent cAMP and glucocorticoid-stimulated transcription. Basal promoter elements and hormone response elements for cAMP, glucocorticoids, and insulin have been defined in previous studies. By using stable transfectants containing a variety of different PEPCK-chloramphenicol acetyltransferase fusion gene constructs, a phorbol ester response sequence, located between positions -437 and -402 relative to the transcription start site, was identified. This region coincides with the insulin response sequence that has recently been defined in the PEPCK promoter. Using a vector containing various wild-type and mutated sequences of this region ligated to the heterologous thymidine kinase promoter, we delineated the boundaries of both elements to the 10 base pairs between positions -416 through -407. Thus, although it has been previously shown that insulin and phorbol esters repress PEPCK gene transcription through distinct pathways, the final target of insulin and phorbol ester action is the same DNA element.