We examined the effects of intracellular Cl- concentration ([Cl-]i) on the epithelial Na channel (ENaC) in a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) with a high rate of Na+ transport produced by stable retroviral transfection with rENaC subunits (Morris RG and Schafer JA. J Gen Physiol 120: 71-85, 2002). Treatment with cAMP (100 microM 8-cpt-cAMP plus 100 microM IBMX) stimulated ENaC-mediated Na+ absorption as well as Cl- secretion via cystic fibrosis transmembrane conductance regulator, which was characterized in alpha-toxin-permeabilized monolayers to have the anion selectivity sequence NO3- > Br- > Cl- > I-. With the use of 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-Isc) 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 6-methoxy-N-(3-sulfopropyl)-quinolinium 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 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 in [Cl-]i from 15 to 145 mM decreased AS-Isc from 24.5 +/- 1.0 to 10.2 +/- 1.6 microA/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 microA/fmol.