We examined the effects of intracellular Cl^- ([Cl^-]_i) on ENaC in a line of MDCK cells (FL-MDCK) with a high rate of Na⁺ transport produced by stable retroviral transfection with rENaC subunits (Morris and Schafer, J Gen Physiol. 120: 71-852, 2002). Treatment with cAMP (100 µM 8-cpt-cAMP plus 100 µM IBMX) stimulated ENaC-mediated Na⁺ absorption as well as Cl^- secretion via CFTR, which was characterized in -toxin-permeabilized monolayers to have the anion selectivity sequence: NO₃^- > Br^- > Cl^- > I^-. Using FL-MDCK monolayers in which the basolateral membrane was permeabilized by nystatin, the ENaC conductance of the apical membrane (determined from the amiloride-sensitive short-circuit current, AS-I_sc, driven by an apical-to-basolateral Na⁺ concentration gradient) was progressively inhibited by increasing the [Cl^-] in the basolateral solution (and hence in the cytosol), but it was insensitive to the [Cl^-] in the apical solution. This inhibitory effect of [Cl^-]_i occurred regardless of the presence or absence of net Cl^- transport. However, from fluorometric measurements using the Cl^--sensitive dye SPQ in intact FL-MDCK monolayers on permeable supports, cAMP, which activates both Na⁺ absorption and Cl^- secretion, produced a decrease of [Cl^-]_i from 76 ± 14 mM to 36 ± 8 mM (P=0.03). Thus it might be expected that activation of Cl^- secretion by cAMP would lead to stimulation rather than inhibition of ENaC. In the nystatin-treated monolayers, an increase of [Cl^-]_i from 15 mM to 145 mM decreased AS-I_s from 24.5 ± 1.0 to 10.2 ± 1.2 µA/cm². This inhibition of ENaC could be attributed to nearly proportional decreases in the density of ENaC in the apical membrane from 1.91 ± 0.16 to 1.32 ± 0.17 fmol/cm² and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 ± 1.2 to 8.2 ± 1.4 µA/fmol.