H4IIE rat hepatoma cells were stably transfected with various phosphoenolpyruvate carboxykinase-chloramphenicol acetyltransferase (PEPCK-CAT) expression vectors. The regulation of the transfected genes was qualitatively similar to that of the endogenous PEPCK gene. CAT expression was increased in response to cAMP and dexamethasone and insulin overrode these effects at concentrations known to be effective in suppressing transcription of the endogenous gene. The effect of insulin was dominant, as it is with the endogenous gene. A series of 5',3', and internal deletions of the PEPCK gene promoter were used to show that this insulin response requires at least two separate elements. One insulin-responsive sequence is located between -468 and -402, relative to the transcription initiation site. The other is between -271 and +69.