Bile pigment gallstones were produced in six of six mongrel dogs by narrowing the cystic duct with ligature after a postoperative interval of seven days. beta-Glucuronidase producing group G Streptococcus and Staphylococcus aureus were found in the bile, the gallbladder wall, and the liver. In another trial similar gallstones were produced in four of eight dogs after mere injection of beta-glucuronidase producing Escherichia coli into the spleen resp. in six of eight dogs after injection of E. coli into the colon without ligature of the cystic duct. In an additional series gallstones were produced in six dogs after injection of beta-glucuronidase producing E. coli into the colon plus ligature of the cystic duct. The injected strain of E. coli was found in the bile, the gallbladder wall, the liver, and even within the produced gallstones. In a control group in none of six dogs gallstones were present. beta-Glucuronidase producing group G Streptococcus was found in the gallbladder of one dog, Pseudomonas aeruginosa was found in the gallbladder of another dog and Staphylococcus aureus in the liver and gallbladder wall of a third dog. We conclude from our experiments that merely bacterial infection of the biliary tract can lead to gallstone formation. The bacterial colonization of the liver and the biliary tract is promoted by biliary tract obstruction. beta-Glucuronidase producing bacteria increase the amount of beta-glucuronidase in the bile thus leading to calcium bilirubinate precipitation and gallstone formation by deconjugation of bilirubindiglucuronide.