The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A and ATP-regulated Cl^-channel that also controls the activity of epithelial Na⁺ channels (ENaC). Previous studies demonstrated that cytosolic domains of ENaC are critical for its down-regulation by CFTR, whereas others suggested a role of cytosolic Cl^-ions. We examined the anion dependence of ENaC and the role of its cytosolic domains for inhibition by CFTR and the Cl^-channel ClC-0. Coexpression of ENaC with CFTR or ClC-0 in Xenopus oocytes inhibited amiloride-sensitive Na⁺ currents after cAMP-dependent stimulation and in presence of a high bath Cl^-concentration without changing surface expression of ENaC. Also injection of Cl^-into oocytes and exposure of inside/out macropatches to high cytosolic Clconcentrations caused inhibition of ENaC. Other anions mimicked the effect of Cl^-. Deletion of the N-terminus of a-, b-or [gamma]ENaC and of the C-terminus of b- and [gamma]ENaC, mutations in the phosphatidylinositol-bisphosphate binding domain of b- and [gamma]ENaC and activation of phospholipase C reduced inhibition by Cl^-. The inhibitory effect of Cl^-was no longer detected in a[Delta]CbENaC channels. These results confirm a role of the C-terminus of bENaC for Cl^-dependent inhibition of the Na⁺ channel, which, however, may only be part of a complex regulation of ENaC by CFTR. Supported by DFG Ku756/7-1