Nuclear extracts from cultured rat hepatocytes were analyzed by gel mobility shift assay for protein binding to the cyclic AMP responsive elements CRE1 (-96/-77) and CRE2 (-152/-132) and the NF1-CTF binding site (-121/-99) of the phosphoenolpyruvate carboxykinase (PCK) promotor. Binding was very weak to the CRE2 and CRE1. The NF1-CTF site formed two complexes with nuclear protein. Protein binding was increased, when the NF1-CTF site was coupled to the CRE1, and further, when it was coupled to both the CRE1 and the CRE2. Complex formation was not altered by treatment of the hepatocytes with glucagon or with glucagon and insulin. Thus, protein binding was most efficient when all three elements were in context, which might be necessary for full transcriptional activation of the PCK gene.