The amiloride-sensitive epithelial sodium channel (ENaC) complex is made up of at least three different subunits alpha, beta, and gamma, which are developmentally regulated, selectively expressed, and variously up-regulated by steroid hormones. To understand mechanisms involved in regulation of the gamma subunit, we have determined the structure of the human gammaENaC gene. By 5' rapid amplification of cDNA ends, primer extension analysis, and nuclease protection assay, we identified transcription start sites in human brain, kidney, and lung. A human genomic library was screened and overlapping cosmid clones that span approximately 50 kilobases and contain the hgammaENaC gene were identified. The 5'-untranslated region is 141 bases long, and the translation start codon is contained within the second exon. The human gene spans at least 35 kilobases. The 5' end of the gene including portions of 5' flanking genomic DNA and the first intron are G + C rich and contain several CpG dinucleotides, consistent with a CpG island. The 5' flanking region contains no CCAAT or TATA-like elements but does contain two GC boxes as well as several putative transcription factor binding sites including AP-2, Sp1, CRE, PEA-3, and NF-IL6. This is the first description of the structural organization and the 5' flanking region of a member of the epithelial sodium channel complex.