The sodium pump, Na,K-ATPase, is an important protein for maintaining intracellular ion concentration, cellular volume, and ion transport and is regulated both transcriptionally and post-transcriptionally. We previously demonstrated that hyperoxia increased Na,KATPase  1 gene expression in Madin-Darby canine kidney (MDCK) cells. In this study, we identify a DNA element necessary for up-regulation of the Na,K-ATPase  1 transcription by hyperoxia and evaluate the nuclear proteins responsible for this up-regulation. Transient transfection experiments in MDCK cells using sequential 5-deletions of the rat Na,K-ATPase  1 promoterluciferase fusion gene demonstrated promoter activation by hyperoxia between ؊102 and ؉151. The hyperoxia response was localized to a 7-base pair region between ؊62 and ؊55, which contained a GC-rich region consistent with a consensus sequence for the SP1 family, that was sufficient for up-regulation by hyperoxia. This GC element exhibited both basal and hyperoxia-induced promoter activity and bound both transcription factors SP1 and SP3 in electrophoretic mobility shift assays. In addition, electrophoretic mobility shift assays demonstrated increased binding of SP1/SP3 in cells exposed to hyperoxia while mutation of this element eliminated protein binding. Other GC sites within the proximal promoter also demonstrated up-regulation of transcription by hyperoxia, however, the site at ؊55 had higher affinity for SP proteins.