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 (V_TE) and transepithelial resistance (R_TE) could be determined. Microelectrodes were used to measure intracellular Cl^- activity (a_i^Cl), basolateral or apical membrane potentials (V_mBL or V_mC) and the voltage divider ratio (Ra/Rb). When a basal cell was impaled, V_mBL was -73±4.3 mV and a_i^Cl 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_i^Cl- of 6.5±0.9 mM. The depolarization and the rate of decrease of a_i^Cl were inhibited by ~60% in the presence of the Cl^- channel blocker flufenamate. Serosal bumetanide significantly decreased the rate of change of a_i^Cl upon removal and readdition of serosal Cl^-. When a luminal cell was impaled VmC was -65±3.6 mV and a_i^Cl was 16.3±2.2 mM. Removal of luminal Cl^- depolarized V_mC and decreased _i^Cl by only 2.5±0.9 mM. Subsequent removal of Cl^- from the serosal bath decreased a_i^Cl in the luminal cell by an additional 6.4±1.0 mM. A plot of V_mBL measurements versus log a_i^Cl/a_o^Cl yielded a straight line (slope=67.8 mV/decade change in aiCl). In contrast, V_mC correlated very poorly with log a_i^Cl/a_o^Cl (slope=18.9mV/decade change in a_i^Cl). These results indicate that: a) in rabbit EEC a_i^Cl is higher than equilibrium across apical and basolateral membranes and this process is independent of bicarbonate, b) the basolateral cell membrane possesses a conductive Cl^- pathway sensitive to flufenamate c) 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^-.