Epithelial Na⁺ channel (ENaC) subunits (α, β and ) found in functional complexes are translated from mature mRNAs that are similarly processed by inclusion of 13 canonical exons. We examined whether individual exons 3 through 12, encoding the large extracellular domain, are required for functional channel expression. Human ENaCs with an in-frame deletion of a single α subunit exon were expressed in Xenopus oocytes and their functional properties were examined by two-electrode voltage clamp. With the exception of exon 11, deletion of an individual exon eliminated channel activity. Channels lacking α subunit exon 11 were hyperactive. Oocytes expressing this mutant exhibited 4-fold greater amiloride-sensitive whole cell currents than cells expressing wild type channels. A parallel 5-fold increase in channel open probability was observed with channels lacking α subunit exon 11. These mutant channels also exhibited a lost Na⁺ self-inhibition, whereas we found similar levels of surface expression of mutant and wild type channels. In contrast, in-frame deletions of exon 11 from either the β or subunit led to a significant loss of channel activity, in association with a marked decrease in surface expression. Our results suggest that exon 11 within the three human ENaC genes encode structurally homologous, yet functionally diverse domains and that exon 11 in the α subunit encodes a module that regulates channel gating.