Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting enzyme of gluconeogenesis in the liver. PEPCK gene expression is controlled at the transcriptional level and is mainly regulated by hormones that are involved in glucose homeostasis. In this study, we have investigated the role of glucose on PEPCK gene expression in cultured hepatocytes. We demonstrate that glucose counteracts the stimulatory effect of glucocorticoids and cAMP on PEPCK expression. Glucose must be metabolized through glucokinase to have its inhibitory effect. The effect of glucose is mainly transcriptional and the region responsible for glucose inhibition is localized in the first 490 bp of the promoter.