Since cholangitis is a major complication in living related liver transplant (LRLT) recipients, rapid detection of biliary bacteria is necessary for the management of patients. We have developed a screening method for the detection of biliary bacteria using flow cytometry (FCM). Two hundred and seventy eight bile samples were obtained from 50 patients with biliary drainage tubes after LRLT at Kyoto University Hospital between July and September 2001. Of the 278 samples, 165 (59.3%) were culture-positive. The most common isolates were Enterococcus species, Pseudomonas species, Staphylococcus species, Klebsiella species, and Candida species. As the original FCM system was inadequate for specifically detecting bacteria in bile samples, we established the most appropriate gate and cut-off value from the particle distribution represented on scattergram of the forward-scattered light and fluorescent light intensity. The 3% cut-off value was most preferably related to the culture results. The FCM system detected biliary bacteria with a sensitivity of 93.9%, specificity of 81.4%, positive predictive value of 88.1%, negative predictive value of 90.2%, false-positive rate of 7.6%, false-negative rate of 3.6%, and percent agreement of 88.9% between FCM and culture. Therefore, FCM can be a useful method in clinical laboratories for the rapid screening for biliary bacteria in LRLT recipients.