Although previous studies have suggested that bacteria may contribute to pigment gallstone formation, the current experiments provide evidence that bacteria have a central role in this process. The studies included scanning electron microscopy (SEM) of gallstones, measurements of bacterial adherence to gallstones in vitro, and determination of glycocalyx elaboration by biliary bacteria. Gallstones from 85 patients were studied under SEM. Twenty-five (78%) of 32 pigment stones had evidence of bacterial microcolonies throughout the interior of the stones. Bacteria were absent from the interior of all 35 cholesterol stones studied. Composite stones (stones with separate pigment and cholesterol portions) showed evidence of bacteria within the pigment portions in 14 (78%) of 18 cases. Biliary bacteria adhered to the surface of pigment gallstones in vitro in 35 (90%) of 39 cases, compared with three (8%) of 39 cholesterol stones. Glycocalyx was elaborated by bacteria isolated from nine (82%) of 11 patients with either pigment or composite gallstones. One (33%) of three bacterial species from patients with cholesterol gallstone disease produced glycocalyx. These studies indicate that most pigment gallstones obtained from patients in Western cultures form as a composite of bacteria, bacterial glycocalyx, and pigment solids. Bacteria were found in the majority of black as well as brown pigment stones. These findings serve as the basis of a new theory of pigment stone formation in which bacteria and glycocalyx are postulated to be responsible for the precipitation and subsequent agglomeration of bilirubin pigment. These results also suggest that sepsis is more common in pigment gallstone disease because the stones can serve as a sanctuary for bacteria.