It has been previously demonstrated that the CCAAT/enhancer-binding protein (C/EBP) trans-activates the gene coding for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) and binds to several sites along the promoter. The additional observations that C/EBP is expressed in liver and follows the same developmental profile as PEPCK suggests that C/EBP plays an important role in the regulation of PEPCK gene expression. However, since C/EBP is expressed at high levels in lung, a tissue in which PEPCK is not expressed, it appears that other mechanisms are involved to provide PEPCK with high level expression in liver. We now show that the albumin promoter D-site-binding protein (DBP), a transcription factor whose expression is limited to the liver, is also able to trans-activate the PEPCK promoter through sequence-specific binding. Both recombinant DBP and C/EBP bind with highest affinity to regions located at positions -85 and -245 in the promoter, but display differences in their binding properties at other sites. Using eukaryotic expression vectors for both C/EBP and DBP, we found that with 5'-deletion mutants of the PEPCK promoter, both C/EBP and DBP exerted their effects through similar regions of the promoter. However, the use of internal deletion mutants of the promoter identified distinct differences in the mechanism of activation by C/EBP and DBP. In particular, a region of the promoter between positions -86 and -117 significantly attenuated the level of trans-activation by DBP, but not by C/EBP. Evidence presented also supports a model whereby the relative ratios of C/EBP and DBP in the cell fine-tune the expression of the PEPCK gene. These results demonstrate that DBP and C/EBP, while having similar DNA binding specificities, have distinct functional differences in the context of the PEPCK promoter. These differences, along with the developmental profiles of C/EBP and DBP, may provide a mechanistic explanation for the liver-specific as well as the developmental profile of PEPCK gene expression.