Beta-glucuronidase activity in the bile may be of importance in the etiology of pigment gallstones. This enzyme is of hepatic or bacterial origin. We have described a method to measure the activity of bacterial beta-glucuronidase in human bile, using 4-nitrophenyl-beta-D-glucopyranosiduronic acid as substrate. The method was used to measure the beta-glucuronidase activity in the bile from 51 patients with gallstone disease. This activity was related to the presence of beta-glucuronidase-producing bacteria in the bile. Escherichia coli, Bacteroides species, and Clostridium perfringens were the only species found to produce beta-glucuronidase. Patients with beta-glucuronidase-producing bacteria had on an average significantly higher enzyme activity in the bile than patients without such bacteria (p less than 0.01). The limitations of using artificial substrates in this type of studies are discussed.