Thyroid hormone (T₃) influences hepatic cholesterol metabolism, and previous studies have established an important role of this hormone in the regulation of cholesterol 7α-hydroxylase (CYP7A), the rate-limiting enzyme in the synthesis of bile acids. To evaluate the respective contribution of thyroid hormone receptors (TR) α1 and β in this regulation, the responses to 2% dietary cholesterol and T₃ were studied in TRα1 and TRβ knockout mice under hypo- and hyperthyroid conditions. Our experiments show that the normal stimulation in CYP7A activity and mRNA level by T₃ is lost in TRβ−/− but not in TRα1−/− mice, identifying TRβ as the mediator of T₃ action on CYP7A and, consequently, as a major regulator of cholesterol metabolism in vivo. Somewhat unexpectedly, T₃-deficient TRβ−/− mice showed an augmented CYP7A response after challenge with dietary cholesterol, and these animals did not develop hypercholesterolemia to the extent as did wild-type (wt) controls. The latter results lend strong support to the concept that TRs may exert regulatory effects in vivo independent of T₃.