The amiloride-sensitive epithelial sodium channel (ENaC) is involved in fluid and electrolyte absorption across a number of epithelia, and cloning of several ENaC subunits has begun to facilitate investigation of the structure, function, and regulation of this channel. Analysis of the amino acid sequence has revealed two potential membrane-spanning domains, but little else is known about the structure of ENaC. To investigate the membrane topology of one subunit, alpha rENaC, we used in vitro transcription, translation, and translocation into microsomal membranes. This generated a glycosylated protein of 93 kDa. Sequence analysis also revealed eight potential sites for N-glycosylation, six of which were found to be glycosylated (Asn190, Asn259, Asn320, Asn339, Asn424, and Asn538), indicating that they are extracellular. The C terminus was localized as intracellular based on antibody recognition and protease sensitivity of a tagged epitope at the C terminus. The N terminus was also found to be intracellular, based on its protease sensitivity. Similar results were obtained by expression in Xenopus oocytes. Together, these results support a model of alpha rENaC consisting of an intracellular N terminus and C terminus, a large N-glycosylated extracellular domain, and two membrane-spanning domains that each pass once through the plasma membrane. Because of their sequence similarity, it is likely that this structure is shared by other ENaC subunits and possibly the degenerins of Caenorhabditis elegans as well.