Iatrogenic bile duct injury during cholecystectomy is the most serious complication of this surgical procedure. Initial reports suggest that this complication is particularly troublesome during laparoscopic cholecystectomy. Proper identification of the biliary anatomy in the subhepatic region is the only way to avoid this catastrophe. The potential benefits of a simple, reliable method for intraoperative delineation of biliary anatomy are self-evident. In this experimental study on rabbits, we show how the simple i.v. injection of a fluorescent bile salt, cholyl-lysyl-fluorescein, enables the surgeon to visualize the entire biliary tree in anatomic detail.