Despite its key role in potassium homeostasis, transcriptional control of the H+-K+-ATPase α2-subunit (HKα2) gene in the collecting duct remains poorly characterized. cAMP increases H+-K+-ATPase activity in the collecting duct, but its role in activating HKα2 transcription has not been explored. Previously, we demonstrated that the proximal 177 bp of the HKα2 promoter confers basal collecting duct-selective expression. This region contains several potential cAMP/Ca2+-responsive elements (CRE). Accordingly, we examined the participation of CRE-binding protein (CREB) in HKα2 transcriptional control in murine inner medullary collecting duct (mIMCD)-3 cells. Forskolin and vasopressin induced HKα2 mRNA levels, and CREB overexpression stimulated the activity of HKα2 promoter-luciferase constructs. Serial deletion analysis revealed that CREB inducibility was retained in a construct containing the proximal 100 bp of the HKα2 promoter. In contrast, expression of a dominant negative inhibitor (A-CREB) resulted in 60% lower HKα2 promoter-luciferase activity, suggesting that constitutive CREB participates in basal HKα2 transcriptional activity. A constitutively active CREB mutant (CREB-VP16) strongly induced HKα2 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. In vitro DNase I footprinting and gel shift/supershift analysis of the proximal promoter with recombinant glutathione S-transferase (GST)-CREB-1 and mIMCD-3 cell nuclear extracts revealed sequence-specific DNA-CREB-1 complexes at −86/−60. Mutation at three CRE-like sequences within this region abolished CREB-1 DNA-binding activity and abrogated CREB-VP16 trans-activation of the HKα2 promoter. In contrast, mutation of the neighboring −104/−94 κβ element did not alter CREB-VP16 trans-activation of the HKα2 promoter. Thus CREB-1, binding to one or more CRE-like elements in the −86/−60 region, trans-activates the HKα2 gene and may represent an important link between rapid and delayed effects of cAMP on HKα2 activity.