Purpose: Cholangiography is often crucial for establishing the definitive diagnosis in infants with prolonged jaundice. Here, we describe our protocol of using the two-hole laparoscopic technique and discuss its benefits. Methods: 144 consecutive patients with suspected biliary atresia were included in this retrospective study. A 5-mm umbilical port is introduced for a 30° laparoscope. An additional 5-mm trocar was inserted at right subcostal incision. A liver biopsy was performed first if needed. The fundus of gallbladder was then exteriorized through the incision of the working port after pneumoperitoneum was released and a catheter is inserted into the gallbladder for cholangiography. Results: The average duration of operation was 34 min (range 20-55 min). Laparoscopic cholangiography failed in 21 cases (14.6%) where atrophic gallbladder was found and BA was confirmed by subsequent laparotomy. For the remaining 123 cases, biliary atresia were diagnosed in 88 (71.5%), biliary hypoplasia in 14 (11.4%), and cholestasis in 21 (17.1%), respectively. There was no bleeding or any other complications intraoperatively. Conclusion: The technique of laparoscopic cholecystocholangiography is simple, safe and efficient. It can provide an accurate diagnosis. Furthermore, for patients without biliary atresia, unnecessary laparotomy can be avoided. © 2010 Springer-Verlag.