Cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, functions as an adenosine 3', 5'-cyclic monophosphate (cAMP)-regulated chloride channel in the apical membrane of biliary epithelial cells, including gallbladder epithelial cells. It has been shown that triangle upF508, the most common CF mutation, impedes CFTR trafficking to the apical surface of epithelial cells. To elucidate the mechanisms of CF biliary disease, we examined structural features, CFTR expression, and chloride transport properties in gallbladder epithelial cells from nine triangle upF508 homozygous liver transplant recipients. Three CF patients had microgallbladders, characterized by severe histological abnormalities. Microgallbladder epithelial cells displayed aberrant immunolocalization of CFTR and of other normally apical proteins in the lateral domain of their plasma membrane and in their cytoplasm. This pattern was mimicked by chronic cholecystitis in non-CF patients. In the 6 remaining CF patients, CFTR was predominantly apical in the gallbladder epithelium, consistent with the detection of a fully glycosylated form by Western blot. In CF as compared with non-CF gallbladder epithelial cells in primary culture, chloride efflux was lower in response to cAMP and tended to be higher in response to exogenous adenosine 5'-triphosphate (ATP). The CF cells exhibited a residual cAMP-dependent chloride secretion that was inversely correlated with ATP-induced chloride secretion, and almost completely blunted in the cells derived from microgallbladders. Our results suggest that epithelial structural alterations aggravate triangle upF508 CFTR mislocalization in the gallbladder epithelium. The associated decrease in residual cAMP-dependent chloride secretion may contribute to biliary damage despite the up-regulation of alternative chloride transport pathways.