The amiloride-sensitive epithelial sodium channel (ENaC) is composed of three subunits: alpha, beta, and gamma. The human alpha-ENaC subunit is expressed as at least two transcripts (N. Voilley, E. Lingueglia, G. Champigny, M. G. Mattei, R. Waldmann, M. Lazdunski, and P. Barbry. Proc. Natl. Acad. Sci. USA 91: 247-251, 1994). To determine the origin of these transcripts, we characterized the 5' end of the alpha-ENaC gene. Four transcripts that differ at their first exon were identified. Exon 1A splices to exon 2 to form the 5' end of alpha-ENaC1, whereas exon 1B arises separately and continues into exon 2 to form alpha-ENaC2. Other variant mRNAs, alpha-ENaC3 and alpha-ENaC4, are formed by activating 5' splice sites within exon 1B. Although alpha-ENaC3 and -4 did not change the open reading frame for alpha-ENaC, alpha-ENaC2 contains upstream ATGs that add 59 amino acids to the previous (alpha-ENaC1) protein. To address the significance of these isoforms, both proteins were expressed in Xenopus oocytes. The cRNA for each alpha-ENaC transcript when combined with beta- and gamma-ENaC cRNA reconstituted a low-conductance ion channel with amiloride-sensitive currents of similar characteristics. We have thus identified variant alpha-ENaC mRNAs that lead to functional ENaC peptides.