The rat amiloride-sensitive epithelial sodium channel (rENaC) is the rate-limiting step for vectorial transport of Na+ across tight epithelia. The complex is composed of three subunits, α, β, and γ. Expression of the subunits has been shown to be tissue-specific and developmentally and hormonally regulated. To study mechanisms involved in transcriptional regulation of αrENaC, we determined the genomic organization of the αrENaC gene. By 5′ rapid amplification of cDNA ends and primer extension, two transcriptional start sites were detected 453 base pairs (bp) apart, resulting in alternative 5′ untranslated region (UTR) lengths of 515 or 62 bp. The longer 5′ UTR is more prevalent in fetal lung than in adult lung or kidney. The 5′ untranslated and coding regions are contained within 12 exons, with the translation start site located within the first exon. Sequence analysis of ∼ 1,500 bp of 5′ flanking DNA identified putative binding sites for transcription factors PEA3, SP1, AP-1, nuclear factor-κB, and thyroid and glucocorticoid receptors. αrENaC promoter-reporter gene constructs produced low levels of reporter gene activity in transiently transfected cells, which could be increased by dexamethasone (DEX) treatment. Tri-iodothyronine treatment alone had no effect but potentiated stimulation by DEX.