OBJECTIVE This study sought to correlate gallbladder (GB) Na+ and Cl-) fluxes with biliary lipid composition during the various stages of gallstone (GS) formation. BACKGROUND GS formation is associated with altered GB ion transport and increased biliary lipid and Ca2+ concentrations. Nonetheless, the longitudinal relationship between ion transport and biliary lipid changes during GS formation has not been defined. METHODS Prairie dogs were fed standard (n = 18) or 1.2% cholesterol-enriched (n = 30) diets for 4 to 21 days. Hepatic and GB bile were analyzed for lipids and Ca2+. Animals were designated either Pre-Crystal, Crystal, or GS based on absence or presence of crystals or GS, respectively. GBs were mounted in Ussing chambers, electrophysiologic parameters were recorded, and unidirectional Na+ and Cl- fluxes measured. RESULTS Short-circuit current and potential difference were similar during Pre-Crystal and Crystal stages but significantly reduced during GS stage compared to controls and Pre-Crystals. Transepithelial resistance was similar in all groups. Net Na+ absorption was increased during Pre-Crystal but decreased during GS stage due to increased mucosa-to-serosa and serosa-to-mucosa flux, respectively. Increased serosa-to-mucosa flux of both Na+ and Cl- characterized the Crystal stage. Biliary lipids and Ca2+ increased progressively during various stages of GS formation and correlated positively with unidirectional fluxes of Na+ and Cl-. CONCLUSIONS GB epithelial ion transport changes sequentially during GS formation, with the early Pre-Crystal stage characterized by increased Na+ absorption, and the later Crystal stage accompanied by prosecretory stimuli on Na+ and Cl- fluxes, which may be due to elevated GB bile Ca2+ and total bile acids.