Microsomal UDP-glucuronosyltransferase activity toward chenodeoxycholic acid and testosterone has been isolated from rat liver and appears to be homogeneous in sodium dodecyl sulfate gel electrophoresis and polyacrylamide gradient gel electrophoresis. The conjugating activities toward chenodeoxycholic acid and testosterone co-purified and showed identical mobilities in disc gel electrophoresis, indicating that chenodeoxycholic acid and testosterone are glucuronidated by a single form of enzyme. UDP-glucuronosyltransferase activities toward estrone, bilirubin, 4-nitrophenol, and morphine did not co-purify with the activity toward chenodeoxycholic acid and testosterone and were not detectable in the pure enzyme in the presence or absence of phospholipids. In addition to glucuronic acid conjugation, the enzyme is able to catalyze galacturonic acid conjugation of chenodeoxycholic acid and testosterone. The enzyme has a subunit Mr approximately 54,000 and in the presence of the stabilizing detergent chenodeoxycholic acid, it appears to exist as an aggregate with an apparent Mr = 318,000 as estimated by gel filtration and 316,000 by polyacrylamide gradient gel electrophoresis.