Recently, several authors have reported that Helicobacter pylori DNA has been found in human bile. The aim of this study is to investigate the presence of H. pylori in the biliary tree of Koreans, including the bile, biliary epithelium, and gallstones. This study analyzed intrahepatic bile, bile duct tissue, and gallstones from 43 patients with hepatobiliary disease (PTCS group), gallbladder bile and tissue from 23 patients with gallbladder disease (CCT group), and eight patients without hepatobiliary disease (control group). H. pylori was examined by PCR with two different primers. PCR was positive in 4/43 (9.3%) by 26 kDa protein antigen primer and in 5/43 (11.6%) by urease A gene primer in bile from the PTCS group. However, in intrahepatic duct tissue, PCR was positive in only one case. PCR of gallbladder bile, tissue, and intrahepatic duct stones was negative. Upon intrahepatic bile analysis, the pH was significantly lower in PCR-positive than in negative cases (P < 0.05). In conclusion, H. pylori DNA may be present in the bile when there are certain environmental changes, such as lowered pH; however, H. pylori does not colonize the bile duct epithelium. We could find no pathogenetic role for H. pylori in the formation of hepatolithiasis.