Tissue responsiveness to a hormone is dependent on the amounts of its receptor expressed under physiological conditions. In the present report, we compared the magnitude of ligand-dependent transactivation mediated by two nuclear hormone receptors, thyroid hormone receptor beta (TR) and retinoid X receptor alpha (RXR), when overexpressed in a variety of cell lines. TR, RXR and reporter (luciferase) genes under the control of artificial hormone response elements were introduced into the cells using recombinant adenovirus (Ad) vectors, to ensure highly efficient gene delivery. Although the amounts of TR expressed were similar in the cell lines infected with Ad-TR, T3 dependent induction of reporter gene expression was significantly greater in HepG2 than in Cos7, GH3, or JEG3 cells, indicating that factors other than TR are limiting the responsiveness to T3. The enhanced response to 9-cis retinoic acid in cells overexpressing RXR was much greater in JEG3 than in HepG2 which had the highest responsiveness to T3 under TR overexpression. These results indicate that the factors affecting T3 responsiveness are not identical to those affecting the 9-cis retinoic acid responsiveness. On the other hand, overexpression of RXR in addition to TR resulted in a decrease in T3-responsiveness in all the cell lines tested, suggesting that some cofactors are common to TR and RXR.