Oxysterols have been detected in various mammalian organs and blood. Biliary epithelium is exposed to high concentrations of cholesterol and we have identified three keto-oxysterols (cholest-4-en-3-one, cholesta-4,6-dien-3-one, cholesta-3,5-dien-7-one) in human bile and gallstones. As the effects of oxysterols on biliary physiology are not well defined, we investigated their biological effects on dog gallbladder epithelial cells. Enriched medium (culture medium containing taurocholate and lecithin and cholesterol ± various oxysterols) was applied to confluent monolayers of dog gallbladder epithelial cells in culture. Cytotoxicity and apoptosis were studied by morphologic analysis and flow cytometry. Oxysterols in the mitochondrial fraction were identified by GC/MS, while release of cytochrome c from mitochondria was assayed by spectrophotometry and Western blot. Compared to cells treated with culture medium or with enriched medium containing cholesterol, oxysterol-treated cells showed significantly increased apoptosis (p<0.05). Exogenously applied oxysterols were recovered from the mitochondrial fraction. Cytochrome c release from mitochondria was increased significantly by cholest-4-en-3-one, cholesta-4,6-dien-3-one and 5-cholestan-3-one (all p<0.05). Thus oxysterols recovered from human bile and gallstones induce apoptosis of biliary epithelium via a mitochondrial-dependent pathway and may play a role in the pathogenesis of chronic inflammation and carcinogenesis in the gallbladder.