Previous studies have identified a region in the promoter of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (positions -460 to +73) containing the regulatory elements which respond to cyclic AMP, glucocorticoids, and insulin and confer the tissue- and developmental stage-specific properties to the gene. We report that CCAAT/enhancer-binding protein (C/EBP) binds to the cyclic AMP-responsive element CRE-1 as well as to two regions which have been previously shown to bind proteins enriched in liver nuclei. The DNase I footprint pattern provided by the recombinant C/EBP was identical to that produced by a 43-kDa protein purified from rat liver nuclear extracts, using a CRE oligonucleotide affinity column, which was originally thought to be the CRE-binding protein CREB. Transient contransfection experiments using a C/EBP expression vector demonstrated that C/EBP could trans activate the PEPCK promoter. The trans activation occurred through both the upstream, liver-specific protein-binding domains and the CRE. The CRE-binding protein bound only to CRE-1 and not to the upstream C/EBP-binding sites. The results of this study, along with physiological properties of C/EBP, indicate an important role for this transcription factor in providing the PEPCK gene with several of its regulatory characteristics.