Epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) are colocalized in the apical membrane of many epithelia. These channels are essential for electrolyte and water secretion and/or reabsorption. In cystic fibrosis airway epithelia, a hyperactivated epithelial Na+ conductance operates in parallel with defective Cl- secretion. Several groups have shown that CFTR down-regulates ENaC activity, but the mechanisms and the regulation of CFTR by ENaC are unknown. To test the hypothesis that ENaC and CFTR regulate each other, and to identify the region(s) of ENaC involved in the interaction between CFTR and ENaC, rENaC and its mutants were co-expressed with CFTR in Xenopus oocytes. Whole cell macroscopic sodium currents revealed that wild type (wt) αβγ-rENaC-induced Na+ current was inhibited by coexpression of CFTR, and further inhibited when CFTR was activated with a cAMP-raising mixture (CKT). Conversely, αβγ-rENaC stimulated CFTR-mediated Cl- currents up to ≃6-fold. Deletion mutations in the intracellular tails of the three rENaC subunits suggested that the carboxyl terminus of the β subunit was required both for the down-regulation of ENaC by activated CFTR and the up-regulation of CFTR by ENaC. However, both the carboxyl terminus of the β subunit and the amino terminus of the γ subunit were essential for the down-regulation of rENaC by unstimulated CFTR. Interesilngly, down-regulation of rENaC by activated CFTR was Cl--dependent, while stimulation of CFTR by rENaC was not dependent on either cytoplasmic Na+ or a depolarized membrane potential. In summary, there appear to be at least two different sites in ENaC involved in the intermolecular interaction between CFTR and ENaC.