Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) can impact thyroid hormone homeostasis in rodents. Increased glucuronidation can result in reduction of serum thyroid hormone and a concomitant increase in thyroid-stimulating hormone (TSH). UGT2B2 is thought to glucuronidate triiodothyronine (T(3)). The purposes of this study were to determine the role of UGT2B2 in T(3) glucuronidation and whether increased T(3) glucuronidation mediates the increased TSH observed after MEI treatment. Sprague Dawley (SD) and UGT2B2-deficient Fischer 344 (F344) rats were fed a control diet or diet containing pregnenolone-16alpha-carbonitrile (PCN; 800 ppm), 3-methylcholanthrene (3-MC; 200 ppm), or Aroclor 1254 (PCB; 100 ppm) for 7 days. Serum thyroxine (T(4)), T(3), and TSH concentrations, hepatic androsterone/T(4)/T(3) glucuronidation, and thyroid follicular cell proliferation were determined. In both SD and F344 rats, MEI treatments decreased serum T(4), whereas serum T(3) was maintained (except with PCB treatment). Hepatic T(4) glucuronidation increased significantly after MEI in both rat strains. Compared with the other MEI, only PCN treatment significantly increased T(3) glucuronidation (281 and 497%) in both SD and UGT2B2-deficient F344 rats, respectively, and increased both serum TSH and thyroid follicular cell proliferation. These data demonstrate an association among increases in T(3) glucuronidation, TSH, and follicular cell proliferation after PCN treatment, suggesting that T(3) is glucuronidated by other PCN-inducible UGTs in addition to UGT2B2. These data also suggest that PCN (rather than 3-MC or PCB) promotes thyroid tumors through excessive TSH stimulation of the thyroid gland.