In airway and renal epithelia, the glucocorticoid-mediated stimulation of amiloride-sensitive Na+ transport is associated with increased expression of the epithelial Na+ channel alpha subunit (alphaENaC). In H441 lung cells, 100 nM dexamethasone increases amiloride-sensitive short-circuit current (3.3 microA/cm2 to 7.5 microA/cm2), correlating with a 5-fold increase in alphaENaC mRNA expression that could be blocked by actinomycin D. To explore transcriptional regulation of alphaENaC, the human alphaENaC 5'-flanking region was cloned and tested in H441 cells. By deletion analysis, a approximately 150-base pair region 5' to the upstream promoter was identified that, when stimulated with 100 nM dexamethasone, increased luciferase expression 15-fold. This region, which contains two imperfect GREs, also functioned when coupled to a heterologous promoter. When individually tested, only the downstream GRE functioned in cis and bound GR in a gel mobility shift assay. In the M-1 collecting duct line Na+ transport, malphaENaC expression and luciferase expression from alphaENaC genomic fragments were also increased by 100 nM dexamethasone. In a colonic cell line, HT29, trans-activation via a heterologously expressed glucocorticoid receptor restored glucocorticoid-stimulated alphaENaC gene transcription. We conclude that glucocorticoids stimulate alphaENaC expression in kidney and lung via activation of a hormone response element in the 5'-flanking region of halphaENaC and this response, in part, is the likely basis for the up-regulation of Na+ transport in these sites.