Glucuronidation of thyroxine (T4) by liver microsomal UDP-glucuronosyltransferase (UDP-GT) is a predominant pathway by which T4 is deactivated. This study was conducted to characterize in vitro T4 UDP-GT activity in rat liver microsomal preparations, to determine if T4 glucuronidation is mediated by a particular form of UDP-GT, and to determine if T4 glucuronidation can be increased by microsomal enzyme inducers. Characterization of microsomal T4 UDP-GT activity led to the establishment of optimal assay conditions. UDP-GT activity toward T4 was determined in hepatic microsomal preparations from Wistar and Gunn rats, a mutant strain of Wistar rats deficient in several forms of UDP-GT. Hepatic microsomal preparations from Gunn rats glucuronidated T4 at one-third the rate catalyzed by microsomal preparations from Wistar rats. To determine the effect of four inducers that each increase a separate class of UDP-GT, phenobarbital (PB), 3-methylcholanthrene (3MC), pregnenolone-16 alpha-carbonitrile (PCN), clofibrate (CLO), saline, or corn oil was administered to male Sprague-Dawley rats ip for 4 days. T4 UDP-GT activity was increased by PB, 3MC, PCN, and CLO 88, 150, 100, and 160%, respectively on a per-milligram-microsomal-protein basis and 138, 125, 100, and 145% on a per-kilogram-body-weight basis, respectively. Therefore, all four classes of UDP-GT inducers increase T4 glucuronidation, suggesting that T4 is not a selective substrate for a particular form of UDP-GT.