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Recently, Helicobacter sp has been identified in resected gallbladder tissue and in collected bile from Chilean patients with chronic cholecystitis. Therefore, it an association between bile Helicobacter sp and gallbladder cancer has been proposed. Interestingly, both Helicobacter colonization and gallstone disease (GD) happen very frequently in Chile. However, whether there is an association between Helicobacter colonization and GD has not been completely studied. The aim of this study was to determine the incidence of Helicobacter in human gallbladder tissues with GD. The study included 95 Mexican patients undergoing cholecystectomy. Collected gallbladder specimens were assessed to identify Helicobacter sp using histology, immunohistochemistry, and polymerase chain reaction (PCR) analysis using Helicobacter-specific 16-S ribosomal RNA primers. Of the 95 specimens examined in detail, all had stones as follows: 56 (59%) had chronic cholecystitis; 7 (7.4%), acute cholecystitis; 15 (16%), both chronic and acute cholecystitis, 10 (9.5%), cholesterolosis, and 7 (7.4%), lymphoid hyperplasia. Specimens were considered positive for Helicobacter when histology was positive. Only 1 of the 95 specimens was positive for Helicobacter by immunohistochemistry analysis; 1 of 32 cases, by PCR. These results suggest a low incidence of Helicobacter in the gallbladder epithelium of Mexican patients with GD. However, we can not discard the existence of uncommon Helicobacter sp in gallbladder epithelium and its association with gallstone pathogenesis. Additionally, this study suggests no apparent association between GD and Helicobacter colonization in a Mexican population.