The enzymatic activity of bacterial beta-glucuronidase plays an essential role in the formation of calcium bilirubinate in bile. There are, however, many unsettled problems such as methodology of the assay for its enzymatic activity. In the present study (1) the azopigments from monoconjugated bilirubin (MCB) and unconjugated bilirubin (UCB) in native bile were semiquantitatively determined, (2) the deconjugation of conjugated bilirubin (CB) in bile was estimated with azopigment analysis and (3) factors affecting the deconjugation of CB in bile, especially for pH value, were investigated. CB in bile was stable at physiologic pH during 6-hr incubation at 37 degrees C, but was hydrolyzed at alkaline pH. At physiologic pH, addition of beta-glucuronidase from E. coli hydrolyzed CB in bile and increased MCB and UCB in bile. Based upon the results mentioned above, it is suggested that alkaline pH and enzymatic activity of beta-glucuronidase should cause the increase of UCB in bile. It can be said that beta-glucuronidase is essential for the formation of calcium bilirubinate gallstone at physiologic pH.