Background. Safe cholecystectomy requires confident identification of extrahepatic biliary anatomy. This is the first report of the use of fluorescein and ultraviolet light to improve visualization of biliary topography during laparoscopic cholecystectomy. Methods. Five patients who had symptomatic gallstones underwent laparoscopic cholecystectomy with intraoperative intravenous fluorescein injection. Ultraviolet A from an LED light source was used to induce fluorescence of bile. It was delivered by a device that was designed and built by the authors. Results. Within 4 to 5 minutes the bile ducts were shining with green fluorescence and were easily differentiated from the surrounding tissues. In all cases, identification of the extrahepatic biliary anatomy by the fluorescence technique preceded its identification with conventional white light. Fluorescence remained for the whole duration of operation that extended for 42 to 77 minutes. Conclusions. At laparoscopic cholecystectomy, intravenous fluorescein injection and ultraviolet A excitation induce bile ducts to fluoresce. The technique allows better and earlier real-time visualization of biliary anatomy than conventional white light. The technique is simple and inexpensive. It serves as an additional tool that would improve safety of laparoscopic cholecystectomy.