We investigated Cl transport pathways in the apical and basolateral membranes of rabbit esophageal epithelial cells (EEC) using conventional and ion-selective microelectrodes. Intact sections of esophageal epithelium were mounted serosal or luminal side up in a modified Ussing chamber, where transepithelial potential difference and transepithelial resistance could be determined. Microelectrodes were used to measure intracellular Cl activity (a), basolateral or apical membrane potentials (V_mBL or V_mC), and the voltage divider ratio. When a basal cell was impaled, V_mBL was 73 ± 4.3 mV and a was 16.4 ± 2.1 mM, which were similar in presence or absence of bicarbonate. Removal of serosal Cl caused a transient depolarization of V_mBL and a decrease in a of 6.5 ± 0.9 mM. The depolarization and the rate of decrease of a were inhibited by ~60% in the presence of the Cl-channel blocker flufenamate. Serosal bumetanide significantly decreased the rate of change of a on removal and readdition of serosal Cl. When a luminal cell was impaled, V_mC was 65 ± 3.6 mV and a was 16.3 ± 2.2 mM. Removal of luminal Cl depolarized V_mC and decreased a by only 2.5 ± 0.9 mM. Subsequent removal of Cl from the serosal bath decreased a in the luminal cell by an additional 6.4 ± 1.0 mM. A plot of V_mBL measurements vs. log a/log a (a is the activity of Cl in a luminal or serosal bath) yielded a straight line [slope (S) = 67.8 mV/decade of change in a/a]. In contrast, V_mC correlated very poorly with log a/a (S = 18.9 mV/decade of change in a/a). These results indicate that 1) in rabbit EEC, a is higher than equilibrium across apical and basolateral membranes, and this process is independent of bicarbonate; 2) the basolateral cell membrane possesses a conductive Cl pathway sensitive to flufenamate; and 3) the apical membrane has limited permeability to Cl, which is consistent with the limited capacity for transepithelial Cl transport. Transport of Cl at the basolateral membrane is likely the dominant pathway for regulation of intracellular Cl.