Common duct gallstones are mainly of the brown pigment type, which are usually attributed to bacterial factors. Bacterial β-glucuronidase most probably plays a role in the pathogenesis in many but not all patients. The role of other bacterial factors is more undecided. The aims of this study were to investigate a possible association between lipopolysaccharides (LPS) and choledocholithiasis, and to examine the interrelationship to β-glucuronidase. Common duct bile obtained at endoscopic retrograde cholangiography in 86 patients was assayed for LPS by a limulus amebocyte lysate test, and β-glucuronidase activity at pH 7.0 was measured. We found that both elevated concentration of LPS and the presence of juxtapapillary duodenal diverticula were associated with common duct stones (p < 0.01, both). Patients who had their common duct stones removed recently had a lower LPS concentration and a lower activity of β-glucuronidase than those who had a stone in situ (p < 0.01 both), but still higher LPS concentration than those without choledocholithiasis at all (p < 0.01). In multiple logistic regression analysis, elevated LPS was the significant predictor of common duct stones (p < 0.01), and not confounding with neither β-glucuronidase nor juxtapapillary diverticula. We conclude that gram-negative bacteria convey bacterial factors associated with choledocholithiasis, by mechanisms independent of, and additional to β-glucuronidase.