Glucocorticoids cause a 10-fold increase in hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription through two low affinity glucocorticoid receptor (GR) binding sites and a complex array of accessory factor DNA elements and associated proteins. To analyze how co-activators interact with the GR in this context, we took advantage of the C656G GR mutant that binds ligand with very high affinity. This GR activates PEPCK gene transcription at a 500-fold lower dexamethasone concentration than does wild type GR. Transfected C656G GR containing additional mutations or deletions was tested on PEPCK gene expression in H4IIE hepatoma cells. We found that the AF2 domain is the only one of the three defined transactivation domains in GR that is required for PEPCK gene expression and that mutation of this domain disrupts the direct interaction of GR with steroid receptor coactivator 1 (SRC-1). These data help define the functional interaction between GR and SRC-1 and further define the role of the GR in glucocorticoid-mediated expression of the PEPCK gene.