Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the initial step in hepatic gluconeogenesis. In the fasted state, PEPCK gene expression is activated by glucagon (via cAMP) and glucocorticoids. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1) plays an important role in energy homeostasis and is considered to be a key regulator of hepatic gluconeogenesis in response to fasting. It is not clear whether PGC-1 is obligatory for the activation of the transcription program of gluconeogenic genes, or whether it amplifies an existing process. H4IIE hepatoma cells were used to address this key point. These cells respond appropriately to all of the hormones involved in the regulation of gluconeogenic genes yet they are devoid of PGC-1. Also, these hormone responses occur in the absence of ongoing protein synthesis, so the necessary complement of transcription factors exists in untreated cells. However, exogenous expression of PGC-1 in these cells does enhance basal and hormone-induced expression of the PEPCK and G6Pase genes. Mutational analyses of the PEPCK gene promoter reveal that one element in the PEPCK gene promoter, gAF3, which binds COUP-TFI, is of particular importance. Taken together, these data suggest that, under chronic fasting conditions, i.e. when high levels of cAMP and glucocorticoids induce PhG-1 expression, this coactivator markedly amplifies PEPCK gene expression and gluconeogenesis.