A morphine UDP-glucuronyltransferase (morphine UGTPB) was purified from liver microsomes of Sprague-Dawley rats treated with phenobarbital. UDP-glucuronyl-transferases in the liver microsomes were solubilized with Emulgen 911 and separated by omega-(beta-carboxypropionyl-amino)octyl Sepharose 4B column chromatography, which has been developed in our laboratory. Morphine UDP-glucuronyltransferases were eluted into two fractions, Peak I and Peak II, which have different substrate specificities. Morphine UGTPB was purified by two times of Chromato-focusing from Peak II which was more specific to morphine. The purified morphine UGTPB gave an apparent pI of 8.0 on chromatofocusing and displayed a subunit molecular weight of 55 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the glucuronidation of 3-hydroxyl group of morphine and small extent of 4-hydroxybiphenyl, but not of androsterone, bilirubin, chloramphenicol, codeine, 4-methylumbelliferone, 4-nitrophenol, testosterone, and 6-hydroxyl group of morphine. The N-terminal amino acid sequences of morphine UGTPB were identical to those of UGT1*01P which is deficient to homozygous Gunn rat. Peak II was absent from the fraction of omega-(beta-carboxypropionylamino)octyl Sepharose 4B column chromatography of liver microsomes of Gunn rats treated with phenobarbital, whereas morphine UGT in Peak I was PB-inducible in Gunn rats. Present results suggest that an isoform of morphine UDP-glucuronyltransferase belongs to the UGT1 family and is phenobarbital-inducible.