To determine whether the isoform of UDP-glucuronosyltransferase which catalyzes the formation of bilirubin monoglucuronide also mediates the formation of bilirubin diglucuronide and other specific sugar conjugates of bilirubin, Wistar rats were treated with clofibrate (300 mg per kg i.p. X 7 days); this resulted in a 200% increase in hepatic transferase specific activity for bilirubin. Proteins from hepatic microsomal fractions were solubilized, and the transferase isoform with activity toward bilirubin was purified by a combination of chromatofocusing, affinity chromatography and hydrophobic chromatography, to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified isoform catalyzed the formation of monoglucuronide and diglucuronide (with UDP-glucuronic acid as a cosubstrate), and glucoside and xyloside (with UDP-glucose and UDP-xylose as respective cosubstrates) of bilirubin and glucuronidation of the carcinogen metabolite 4'-hydroxydimethylaminoazobenzene. It also catalyzed the conversion of bilirubin monoglucuronide to diglucuronide (with UDP-glucuronic acid as cosubstrate, pH optimum 7.8), to mixed glucuronide-glucoside conjugate (with UDP-glucose as a cosubstrate) and to unconjugated bilirubin (with UDP as a cosubstrate, pH optimum 5.5). Each transferase activity was copurified at each purification step. Results of enzyme kinetic studies suggest that UDP-glucuronic acid, UDP-glucose and UDP-xylose recognize a common site. Transferase activities toward bilirubin were not detectable in homozygous Gunn rats liver microsomal fractions; in heterozygous Gunn rats, these activities were reduced by 40 to 60%. The results suggest that conjugation of bilirubin with glucuronic acid, glucose or xylose is catalyzed by a single transferase isoform.