Renal prostaglandins modulate the activity of a number of the transport systems in the kidney, including the Na,K-ATPase. Not only do prostaglandins have acute affects on renal Na,K-ATPase, but in addition prostaglandins have chronic affects, which include regulation at the transcriptional level. Previously, we have presented evidence that one such prostaglandin, PGE1, stimulates the transcription of the human Na,K-ATPase 1 subunit gene in MDCK cells via the cAMP, Protein Kinase C (PKC) and Ca²⁺ mediated pathways. Evidence was presented indicating that the PGE₁ stimulation was mediated through the binding of cyclic AMP Regulatory Element Binding Protein (CREB) to a Prostaglandin Responsive Element (PGRE), as well as Sp1 binding to an adjacent Sp1 site. In this report we present evidence from Electrophoretic Mobility Shift Assays (EMSAs) and DNA affinity precipitation studies, that another PGRE present in the Na,K-ATPase 1 subunit promoter similarly binds CREB, and Sp1. The evidence which indicates a requirement for CREB as well as Sp1 for gene activation through both PGREs (PGRE1 and PGRE3) includes studies with a dominant negative CREB (KCREB), Drosophila SL2 cells and PGRE mutants. The results of these studies are indicative of a synergism between Sp1 and CREB in mediating regulation by PGRE3, while regulation occurring through PGRE1 also involves Sp1 and CREB, the mechanism appears to be distinct.