The molecular pathogenesis of cystic fibrosis (CF) liver disease is unknown. This study investigates its earliest pathophysiologic manifestations employing a mouse model carrying F508, the commonest human CF mutation. We hypothesized that, if increased bile salt spillage into the colon occurs as in the human disease, this should lead to a hydrophobic bile salt profile and to "hyperbilirubinbilia" because of induced enterohepatic cycling of unconjugated bilirubin. Hyperbilirubinbilia may then lead to an increased bile salt to phospholipid ratio and, following hydrolysis, precipitation of divalent metal salts of unconjugated bilirubin. We document in CF mice elevated fecal bile acid excretion and biliary secretion of more hydrophobic bile salts compared with control wild type mice. Biliary secretion rates of bilirubin monoglucuronides, bile salts, phospholipids and cholesterol are increased significantly with an augmented bile salt to phospholipid ratio. Quantitative histopathology of CF livers displays mild early cholangiopathy in 53% of mice and multifocal divalent metal salt deposition in cholangiocytes. We conclude that increased fecal bile acid loss leads to more hydrophobic bile salts in hepatic bile and to hyperbilirubinbilia, a major contributor in augmenting the bile salt to phospholipid ratio and endogenous -glucuronidase hydrolysis. The confluence of these perturbations damages intrahepatic bile ducts and facilitates entrance of unconjugated bilirubin into cholangiocytes. This study of the earliest stages of CF liver disease provides a framework for investigating the molecular pathophysiology of more advanced disease in murine models and in humans with CF.