Prolonged dietary Na+ depletion and chronic administration of adrenal steroids increase steady-state mRNA levels of the gamma subunit of the epithelial sodium channel (gammaENaC) in rat colon. This increase correlates with a marked increase in transepithelial Na+ transport and is thought to occur via transcriptional regulation. To begin to evaluate these mechanisms in detail, we determined the organization of the rat gammaENaC gene. A rat genomic library was screened and overlapping lambda clones that together span the gene (approximately 36 kb) and contain at least 1 kb of 5' flanking genomic DNA were isolated. As in the human gene, the rat gammaENaC gene contains 13 exons and a CpG island at the 5' end of the gene. A single transcription start site was identified in rat kidney by nuclease protection assay defining a 5' untranslated region of 126 nt. The translation initiation codon was identified within the second exon and the entire 3' UTR (approximately 1 kb) was within the last exon. 800 bp of 5' flanking sequence, as well as the 3.4 kb first intron, were sequenced and analyzed for transcriptional regulatory motifs. Analogous to the human gammaENaC gene [Thomas, C.P., Doggett, N.A., Fisher, R., Stokes J.B., 1996. Genomic organization and the 5' flanking region of the gamma subunit of the human amiloride-sensitive epithelial sodium channel. J. Biol. Chem. 271, 26 062--26 066], two GC boxes were seen at -30 and -61 to the transcription start site. In addition, putative AP-1, AP-2, CRE, Sp1 and GATA-1 and GRE motifs were identified elsewhere in the 5' flanking region or the first intron. Two mammalian-wide interspersed repeats and a rodent-specific B1 repeat were also identified within the first intron. Fragments containing the putative GRE motifs coupled to luciferase did not confer a glucocorticoid-stimulated response in two cell lines that contained a functional glucocorticoid receptor. However, a 76 nt rat gammaENaC 5' flanking fragment (-76 to +68) directed expression of luciferase in the epithelial cell lines H441 and FRTL5, suggesting that this minimal region that contained both GC boxes was sufficient for promoter activity.