AIM:To search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture.METHODS:DNA was extracted from cholesterol gallstones in gallbladders and nested primers polymerase chain reaction (NP-PCR) was used to amplify bacterial gene fragments for identifying the existence of bacteria. The samples of bacterial DNA extracted from potentially causative or unrelated living bacteria were amplified in vitro as the standard markers and comparative 16S ribosomal RNA sequence analysis was made for bacterial identification.RESULTS:The gallbladder gallstones of 30 patients were analyzed and bacterial DNA was found in 26 patients. Among them, gallstones with cholesterol content between 30%-69% were seen in 5 (5/5) patients, 70%-90% in 11(11/14) patients, and more than 90% in 10(10/11) patients. There was no difference either in cholesterol and water content of gallstones or in harboring bacterial DNA of gallstones.E.coli-related DNA fragments appeared in the stones of 8 (26.67%) patients; propionibacteria type DNA in 7 (23.33%); and harbored bacterial gene fragments in 2 patients, similar to Streptococcus pyogenes. A more heterogenous sequence collection was found in 7 (23.33%) patients, which could belong to multiple bacterial infections.Two (6.67%) patients had bacterial DNA with low molecular weight which might be related to some unidentified bacteria.CONCLUSION:Most cholesterol gallstones harbor bacterial DNA.It is important to determine whether these microorganisms are innocent bystanders or active participants in cholesterol gallstone formation.