Ezetimibe [1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone] (Zetia; Schering-Plough, Kenilworth, NJ) is the first in a new class of cholesterol-lowering agents known as cholesterol absorption inhibitors. The objective of this study was to identify the isoform(s) of human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for the glucuronidation of ezetimibe. The main circulating metabolite of this drug in human plasma is SCH 60663, the phenolic glucuronide conjugate of ezetimibe. SCH 60663 [m/z = 584 Thompsons (Th)] is also the major in vitro metabolite formed by human liver microsomes supplemented with UDP glucuronic acid (UDPGA). In contrast to the liver, human jejunum microsomes supplemented with UDPGA converted ezetimibe to two glucuronides with the same mass (m/z = 584 Th) by liquid chromatography-mass spectrometry. One corresponds to the phenolic glucuronide (1-O-[4-trans-2S,3R)-1-(4-fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4-fluorophenyl)propyl]-2-azetidinyl]phenyl-beta-D-glucopyranuronic acid; SCH 60663) and the other was identified as the benzylic glucuronide of ezetimibe (1-O-[1(S)-(4-fluorophenyl)-3-[1-(4-fluorophenyl)-2(S)-(4-hydroxyphenyl)-4-oxo-3(R)-azetidinyl]propyl]-beta-D-glucopyranuronic acid; SCH 488128). Recombinant human UGT1A1, UGT1A3, and UGT2B15 all exhibited catalytic activity with respect to the formation of the phenolic glucuronide. However, UGT2B7 exclusively formed SCH 488128, a trace metabolite detected in dog and human plasma samples after oral administration of ezetimibe. In conclusion, the formation of SCH 60663 is mediated via UGT1A1, UGT1A3, and UGT2B15, and the formation SCH 488128 is mediated via UGT2B7.