The amiloride-sensitive epithelial Na⁺ channel (ENaC), found in the apical membrane of Na⁺-absorptive epithelia, is made up of three differentially regulated subunits: , , and . We undertook a study of the 5'-end of the gene encoding the -ENaC subunit in the rat. 5'-Rapid amplification of cDNA ends and RNase protection assays indicated multiple transcription start sites over a 50-bp region. Sequencing 1.3 kb of the 5'-flanking DNA revealed putative binding sites for PEA3, Sp1, activator protein (AP)-1 and Oct-1 but neither a TATA box nor consensus sites for steroid hormone receptor binding. Transient transfections of reporter constructs driven by -ENaC 5'-flanking DNA in the representative epithelial cell lines Madin-Darby canine kidney, MLE-15, and Caco-2 revealed a negative element present between positions 424 and 311 that affected basal transcription rates. Gel shift assays showed protein-DNA binding activity of an AP-1 consensus site in this region; however, mutation of the AP-1 site did not abrogate the repressive activity of the region in transient transfections. Deletion of two clusters of Sp1 consensus binding sites between 1 and 51 bp and between 169 and 211 bp indicated that the proximal cluster was essential to basal promoter activity in transfected cell lines. In a comparison of these data with those in published studies on - and -ENaC promoters, the - and -subunit promoters appear to be more similar to each other than to the -promoter.