Despite its key role in potassium homeostasis, transcriptional control of the H⁺-K⁺-ATPase 2 (HK2) gene in the collecting duct remains poorly characterized. cAMP increases H⁺-K⁺-ATPase activity in the collecting duct, but its role in activating HK2 transcription has not been explored. Previously, we demonstrated that the proximal 177 bp of the HK2 promoter confers basal collecting duct-selective expression. This region contains several potential cAMP response elements (CREs). Accordingly, we examined the participation of CRE binding protein (CREB) in HK2 transcriptional control in mIMCD3 cells. Forskolin and vasopressin induced HK2 mRNA levels, and CREB overexpression stimulated the activity of HK2 promoter-luciferase constructs. Serial deletion analysis revealed that CREB inducibility was retained in a construct containing the proximal 100 bp of the HK2 promoter. In contrast, expression of a dominant-negative inhibitor (A-CREB) resulted in 60% lower HK2 promoter-luciferase activity, suggesting that constitutive CREB participates in basal HK2 transcriptional activity. A constitutively active CREB mutant (CREB-VP16) strongly induced HK2 promoter-luciferase activity, whereas overexpression of CREB-dLZ-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 GST-CREB-1 and miMCD3 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 HK2 promoter. In contrast, mutation of the neighboring -104/-94 B element did not alter CREB-VP16 trans-activation of the HK2 promoter. Thus, CREB-1, binding to one or more CRE-like elements in the -86/-60 region, trans-activates the HK2 gene and may represent an important link between rapid and delayed effects of cAMP on HK2 activity.