Chloride is the major inorganic anion in bile but its mechanism of passage from blood to bile is uncertain. Specific membrane channels account for most net inorganic anion flux in other cell types such as the proximal tubular cell and red blood cell; disulfonic stilbenes inhibit anion movement through these channels. Therefore, we have sought the presence of similar channels in the hepatocyte. Net inorganic anion flux or conductance was initiated in isolated rat hepatocytes by valinomycin in the presence of an outward potassium gradient. Potassium concentration in the extracellular medium increased from 2.75 ± 0.02 in control cell suspensions to 3.15 ± 0.04 in valinomycin-treated cell suspensions. Membrane potential difference (Em) (mV), determined as the distribution of [14C]tetraphenyl phosphonium ion was −28 mV in control cells and −42 mV in valinomycin-treated cells (p < 0.05). Intracellular chloride concentration (36Cl−) (mEq per liter of cell water) decreased significantly from 38.6 in control cells to 32.0 in valinomycin-treated cells. The observed intracellular concentration (36Cl−) in both control and valinomycin-treated cell suspensions closely approximates values predicted on the basis of the Nernst equation: 41 and 29 (mEq per liter of cell water), respectively, suggesting that the chloride ion is passively distributed on the basis of the membrane potential difference. Furthermore, net rate-limited cell water loss of approximately 15% of control values was associated with the above valinomycin-stimulated changes in ion distribution, as assessed using three methods of cell water volume determination. 4,4′-Diisothiocyanostilbene-2,2′-disulfonate (DIDS), a specific inorganic anion channel blocker, inhibited the valinomycin-induced changes in chloride ion distribution; DIDS (0.8 mM-treated cells contained 45.2 ± 4.4 mEq per liter (36Cl−) following hyperpolarization with valinomycin whereas untreated cells contained 33.3 ± 4.8 mEq per liter of cell water after valinomycin treatment (p < 0.05). Preincubation with DIDS (0.8 mM) also significantly suppressed valinomycin-induced changes in external K+ concentration and cell water volume. These observations are consistent with the hypothesis that a channel exists for the electrogenic transport of chloride ion in isolated rat hepatocytes.