Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is regulated by a variety of agents. Glucocorticoids, retinoic acid, and glucagon (via its second messenger, cAMP) stimulate PEPCK gene transcription, whereas insulin, phorbol esters, cytokines, and oxidative stress have an opposing effect. Stimulation of PEPCK gene expression has been extensively studied, and a number of important DNA elements and binding proteins that regulate the transcription of this gene have been identified. However, the mechanisms utilized to turn off expression of this gene are not well-defined. Many of the negative regulators of PEPCK gene transcription also stimulate the nuclear localization and activation of the transcription factor NF-kappa B, so we hypothesized that this factor could be involved in the repression of PEPCK gene expression. We find that the p65 subunit of NF-kappa B represses the increase of PEPCK gene transcription mediated by glucocorticoids and cAMP in a concentration-dependent manner. The mutation of an NF-kappa B binding element identified in the PEPCK gene promoter fails to abrogate this repression. Further analysis suggests that p65 represses PEPCK gene transcription through a protein protein interaction with the coactivator, CREB binding protein.