Induction of UDP-glucuronosyltransferases (UGTs) toward thyroxine (T4) and p-nitrophenol (pNP) by 3-methylcholanthrene (MC), dexamethasone (DEX), clofibrate (Cl), and MC combined with DEX or Cl was studied in rat hepatocyte culture. We have developed a sensitive method for the measurement of glucuronide conjugates of the two substrates based on HPLC analysis of culture medium. MC, Cl, or DEX increased the activity of T4 UGT. Combination of MC and Cl showed additive effect, enzyme activity was enhanced compared with either MC or Cl treatment alone (617, 441, and 217% of the control, respectively). Combination of MC and DEX did not result in higher T4 UGT activity than MC treatment alone. Both MC and DEX enhanced the pNP UGT activity (182 and 162% of the control, respectively). Combination of MC with DEX resulted in additive effect. Cl treatment did not affect pNP conjugation either alone or in combination with MC. Western blot analysis revealed that only the amount of UGT1A1 was elevated by Cl and DEX. In contrast, concentration of UGT1A6 was increased by MC. Previous studies demonstrated that UGT1A1 inducers like phenobarbital have no effect on T4 conjugation (). Our results suggest that Cl, a known inducer of UGT1A1, enhances the activity of other enzyme(s) involved in T4 glucuronidation as well. It is well documented that DEX potentiates the inductory effect of polycyclic aromatic hydrocarbon on UGT1A6 (). In our study, MC increased the rate of T4 glucuronidation, and DEX had no additional effect on this reaction, suggesting that UGT1A6 is not the only enzyme inducible by MC that can catalyze T4 conjugation.