PEPCK transcription is induced by cAMP/PKA and glucocorticoids (Dex) and is inhibited by insulin to regulate blood glucose. Recent reports suggested that C/EBP binding to the PEPCK CRE plays a role in Dex induction and that insulin-induces inhibitory forms of C/EBP to inhibit transcription. Here, we assessed the roles of CREB and C/EBP factors in mediating hormone-regulated transcription. Neither cAMP nor insulin regulated the phosphorylation of C/EBP. Cycloheximide did not block insulin inhibition, indicating that alternate translation of C/EBP is not required. Dominant negative CREB or C/EBP blocked induction by PKA, but neither affected regulation by Dex or insulin. Tethering the activation domains of CREB or C/EBP to a CRE-> Gal4 site mediated varying extents of basal and PKA inducible activity, but neither activation domain affected induction by Dex or inhibition by insulin. Surprisingly, synergistic induction by PKA and Dex did not require the CRE and was unaffected by dominant negative CREB or C/EBP. PKA and Dex also synergistically induced a minimal 3XGRE promoter, but inhibited Dex induction of the MMTV and IGFBP-1 promoters, even though PKA alone did not regulate these promoters. These results suggest that PKA modifies the activity of other factors involved in Dex induction to mediate synergistic induction or inhibition in a promoter-specific manner. Our data indicate that the roles of CREB and C/EBP are restricted to mediating PEPCK induction by PKA, and that other factors mediate PEPCK induction by Dex, synergism between PKA and Dex, and inhibition by insulin.