Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the rate limiting step in hepatic and renal gluconeogenesis. Glucagon (acting via cyclic AMP (cAMP)) and glucocorticoids stimulate PEPCK gene transcription, whereas insulin has the opposite effect. Since these are the major regulatory hormones controlling glucose homeostasis, and because increased hepatic glucose production is one of the characteristics of non-insulin dependent diabetes mellitus (NIDDM), investigators have speculated that the regulation of PEPCK gene expression may be defective in patients with NIDDM. To begin to investigate this possibility we have isolated and sequenced the human PEPCK gene promoter. In addition, we have constructed and analyzed a human PEPCK promoter-chloramphenicol acetyltransferase (CAT) fusion gene in an effort to correlate differences between the rat and human promoter sequences and the hormonal regulation of transcription.