Background: It has been reported that several species of Helicobacter colonize the biliary tract of animals and human, but their participation in hepatobiliary diseases are not established. This study is undertaken to determine if Helicobacter genus members, especially with regard to H. pylori, could be detected in the bile and gallbladder diseases. Methods: Thirty-eight gallbladder tissues and thirty-six bile samples recovered from 38 patients with different gallbladder diseases were subjected to rapid urease test, culture and an established Multiplex-PCR using two pairs of primers based on 16s rRNA and isocitrate dehydrogenase genes specific for Helicobacter genus and H. pylori species, respectively. Bile and tissue samples from 40 autopsy gallbladders with normal pathology were tested by PCR as control group. Results: In 4 of 36 bile samples H. pylori was identified using PCR. None of the tissue samples of the patients and controls were positive in PCR for Helicobacter DNA. We were not successful in isolation of Helicobacter using culture method. Conclusion: This study showed the presence of H. pylori DNA in the bile samples of a small sample size of patients with biliary diseases in south of Iran. We could find no pathogenetic role for H. pylori in the formation of hepatobiliary disease. To establish a clinical role for Helicobacter species in the hepatobiliary diseases, more studies on a larger group of patients and control groups are needed to ascertain whether Helicobacter species or their component might have a role as a causative agent or cofactor in the pathogenesis of biliary tract diseases in human.Iran J Med Sci 2006; 31(4): 186-190.