Epithelial sodium channels (ENaC) are assembled in the endoplasmic reticulum (ER) from , and subunits, each with two transmembrane domains, a large extracellular loop and cytoplasmic amino and carboxyl termini. ENaC maturation involves transit through the Golgi complex where Asn-linked glycans are processed to complex type and the channel is activated by furin-dependent cleavage of the and subunits. To identify signals in ENaC for ER retention/retrieval or ER exit/release, chimera were prepared with the interleukin subunit (Tac) and each of the three cytoplasmic carboxyl termini of mouse ENaC (Tac-Ct) or with -glutamyltranspeptidase and each of the three cytoplasmic amino termini (Nt-GGT). By monitoring acquisition of endoglycosidase H resistance after metabolic labeling, we found no evidence of ER retention of any chimera when compared to control Tac or GGT, but we did observe enhanced exit of Tac-Ct when compared to Tac. ER exit of ENaC was assayed after metabolic labeling by following the appearance of cleaved , as cleaved subunit, but not non-cleaved , is endoglycosidase H resistant. Interestingly, ER exit of epitope-tagged and truncated (delta624-699-V5) with full-length was similar to wild type (+), whereas ER exit of ENaC lacking the entire cytoplasmic carboxyl tail of (delta613-699-V5 +) was significantly reduced. Subsequent analysis of ER exit for ENaCs with mutations within the intervening sequence H⁶¹³RFRSRYWSPG⁶²³ within the context of the full-length , revealed that mutation RSRYW⁶²⁰/AAAAA significantly reduced ER exit. These data indicate that ER exit of ENaC is regulated by a signal within the subunit carboxyl cytoplasmic tail.