The multiple indicator dilution technique was used to study transfer of labeled HCO3∼ across the hepatocyte membrane in the anesthetized mongrel dog. A bolus of H['4C]O3∼, JICr-labeled erythrocytes, [36CI−] and/or [3H]sucrose, and [3H]OH was injected through a catheter in the portal vein, and timed anaerobic blood samples were obtained from a catheter in the hepatic vein. Experiments were carried out in untreated controls and after intravenous infusion of acetazolamide (100 mg/kg). In the controls, the H[14C]O3curve was very similar to the [3H]OH curve. The dilution curves were all linear transformations of each other, indicating that HCO3∼, as had previously been shown for the other diffusible tracers, undergoes delayed-wave flow-limited distribution. The distribution space for H[14C]O3∼ in the control situation includes the blood plasma and interstitial spaces, the erythrocyte interior modified by a Donnan equilibrium, and the available liver cellular space. The calculated HCO3∼ concentration in the liver cells was somewhat lower than that in the plasma space; the difference implied a cellular pH lower than that of plasma by approximately 0.08 pH units. When the carbonic anhydrases were inhibited with acetazolamide, the dilution curve for H[HC]O3∼ changed radically, approaching that for [36Cl], which does not enter the liver cells. The change indicates that although HCO3∼, like Cl", is rapidly exchanged between plasma and erythrocytes, it also does not readily penetrate hepatocytes unless previously transformed to carbon dioxide by the carbonic anhydrases.