The hormonal regulation of transcription of the phosphoenolpyruvate carboxykinase (GTP) (4.1.1.32) (PEPCK) gene during diabetes was studied using transgenic mice containing a chimeric gene consisting of segments of the PEPCK promoter (-2000/+73, -460/+73, -355/+73) linked to bovine growth hormone (bGH) reporter gene. The effect of diabetes and insulin on transgenic mice containing a mutation in cAMP regulatory sequences at -90/-82 and -250/-234 was also studied. In addition, we analyzed the transcriptional response of the PEPCK gene to adrenalectomy, the administration of glucocorticoids, and alterations in dietary protein and carbohydrate. Our results indicate that deletion of the insulin regulatory sequence of the PEPCK promoter did not affect dietary control of PEPCK gene expression. However, glucocorticoids and the glucocorticoid regulatory unit appear to be essential for induction of PEPCK gene transcription by diabetes. By contrast, mutation of cAMP regulatory elements of the PEPCK promoter did not limit induction of PEPCK transcription by diabetes, nor did it affect negative regulation of transcription by insulin. These results provide evidence for the interaction of insulin and glucocorticoid regulatory elements in the control of PEPCK gene transcription and suggest an important role of glucocorticoids as a gluconeogenic activator during diabetes.