Bacteria are often found in high concentrations in brown pigment and less so in cholesterol gallstones. Although it is intriguing to hypothesize that cholesterol stone formation is non-bacterial in nature and principally different from the pathogenesis of "infectious" brown pigment gallstones, it is more likely that significant overlap exists between the two processes. Most gallstones are composite in nature. Using molecular-genetic methods, bacteria can be found in most pure cholesterol gallstones (i.e. those whose structure consists of more than 90% cholesterol). The natural history of the gallstones development is unknown. It is likely that brown pigment stones can evolve in their chemical composition after the termination of the infectious process that initiate their formation, and may further develop into either mixed or nearly pure cholesterol stones. In a similar fashion, cholesterol-poor or black pigment gallstones may act as foreign bodies to enhance the propensity of bacterial colonization in the presence of pre-existing gallstones or cholangitis, thereby activating pathways of bacterial lithogenesis and resulting in the encasement of cholesterol nuclei with pigment shells and/or in the internal remodeling of extant stones. It is often difficult, if not impossible, to ascertain whether bacterial infection of bile arose before stone formation or vice-versa. The development of gallstones (nucleation, assembly of microcalculi, growth, remodeling) includes the interaction of both bacterial and non-bacterial mechanisms, working discontinuously over years and decades and shaping the structural individuality of each stone. At cholecystectomy, the gallstone removed from the patient represents the end product of a long pathologic process. Although our understanding of the exact temporal contribution of bacteria in lithogenesis is incomplete, it is important for the clinician to realize that most gallstones are colonized by a bacterial biofilm, even though the bile may be culture-negative.