The aim of the this study was to search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture by NP-PCR technique. Bacterial gene fragments were amplified in vitro from DNA which were extracted from cholesterol gallstones in gallbladder for identifying the existence of bacteria. The gallbladder gallstones of 30 patients were analysed. Bacterial DNA was found in the stones of 26 patients, indicating that most cholesterol gallstones harbor bacterial DNA.