To study how ursodeoxycholic acid (UDCA) increases biliary HCO3- concentration and alkalinizes bile, intracellular pH (pHi) and canalicular pH (pHc) were measured microfluorimetrically in isolated rat hepatocyte couplets (IRHC). Isolated perfused rat livers (IPRL) were also used to assess the roles of Cl-, HCO3-, and zone III hepatocytes. In IRHC, UDCA diminished pHi only when HCO3- was omitted. pHi recovery was inhibited by amiloride. UDCA did not affect pHi recovery from an acid load (NH4Cl) nor modify pHc (+HCO3-). In IPRL, biliary HCO3- concentration increased following UDCA despite removal of Cl- (to inhibit Cl(-)-HCO3- exchanger) or destruction of zone III hepatocytes with digitonin. Moreover, when HCO3- was omitted from the perfusate, biliary pH rose following UDCA even though the hypercholeresis was abolished. Thus 1) hepatic UDCA uptake represents an acid load that is counteracted by Na(+)-H+ exchange when HCO3- is absent; 2) UDCA does not alkalinize pHc; and 3) alkalinization of biliary pH in IPRL is not HCO3- dependent, does not involve Cl(-)-HCO3- exchange, or zonal differences in UDCA metabolism or excretion. UDCA appears to alkalinize bile by protonation within the bile duct lumen. UDCAH may then cross the biliary epithelium.