Recent studies in perfused livers and isolated hepatocytes indicate that ursodeoxycholic acid-induced HCO3-rich hypercholeresis originates at the ductule/duct level. The bile duct epithelium may be involved in bile alkalinization by passively reabsorbing the protonated unconjugated ursodeoxycholic acid, by directly secreting in response to an ursodeoxycholic acid-induced increase in acid/base transporter activity or by taking up UDCA in exchange for a base equivalent. To investigate these processes in more detail, we studied the effects of ursodeoxycholic acid on intracellular pH in SK-ChA-1, a well-differentiated human cholangiocarcinoma cell line similar to bile duct epithelium in terms of intracellular pH regulatory mechanisms and morphological markers. Intracellular pH changes were monitored with a microfluorimetric setup using the fluorescent indicator 2′ −7′-bis(2-car-boxyethyl)−5, 6, carboxy fluorescein. Administration of 50 to 1,000 μmol/LUDCA in the absence of HCO3 caused dose-dependent intracellular acidification (intracellular pH = −0.13 ± 0.03 pH/U after 500 μmol/L ursodeoxycholic acid). Acidification was not prevented by preincubation of cells with 0.5 mmol/L 4,4-diisothiocy-anatostilbene-2,2,-disulfonic acid (DIDS) for 30 min or by furosemide administration (1 mmol/L), thus ruling out the stimulation of Cl/HCO3 exchange or the presence of an ursodeoxycholic acid/base exchange. Ursodeoxycholic acid also acidified human fibroblasts, a cell type with no transport capability for ursodeoxycholic acid. In addition, direct measurement of the activities of the three major acid/base transporters in SK-ChA-1 cells (Na+/H+ exchange, sodium-dependent and sodium-independent Cl/HCO3 exchange) failed to show significative differences between cells treated with 500 μmol/L UDCA and controls. In conclusion, ursodeoxycholic acid administration does not primarily stimulate the activities of acid/base transporters responsible for HCO3 secretion by bile duct epithelium. Rather, similar to what has previously been shown in hepatocytes, ursodeoxycholic acid induces intracellular acidification in SK-ChA-1 cells and in fibroblasts. This effect is likely mediated by nonionic diffusion of the weak acid ursodeoxycholic acid. These data confirm that ursodeoxycholic acid can be passively reabsorbed by the biliary epithelium, consistent with the cholehepatic-shunt hypothesis. (Hepatology 1994;19:145–154).