The role of bacteria in gallstone formation could not be conclusively evaluated until bacterial presence or absence in a stone was consistently shown. Cultural bacteriologic investigations at the time of cholecystectomy, however, led to the assumption that cholesterol gallstones were free of bacteria. In this study, we used a culture independent, molecular genetic approach to detect, quantify, and identify bacteria in cholesterol gallstones from 100 patients at the time of cholecystectomy and 6 months following. Bacterial growth was recorded in the culture in 9 of 100 gallstones; bacterial DNA, however, was detected in 82 of 91 sterile gallstones. High concentrations corresponding to between 10(6) to 10(7) bacteria/g were detected in 11 stones and low concentrations of 10(5) bacteria/g were detected in 71 sterile stones. The infection in stones with a positive bacterial culture was characterized by the predominance of single bacterial sequence(s) of the bacteria cultured. A similar predominance, indicating a recent infection, was found in sterile gallstones with low DNA concentrations. A high diversity of non-repeating bacterial sequences, possibly arising from previous overlapping infections, was found in sterile gallstones with high concentrations of bacterial DNA. After 6 months concentrations of bacterial DNA fell significantly in all groups of gallstones. As bacterial DNA is quickly destroyed upon storage, but is nevertheless readily found in most gallstones at the time of cholecystectomy, there must be a mechanism by which it is replenished. One such mechanism is the frequently reoccurring, possibly self-terminating infection and another one is the permanent colonization of the gallstone with bacteria at low concentrations. Both can promote cholecystolithiasis.