T3 potently influences cholesterol metabolism through the nuclear thyroid hormone receptor beta (TRbeta), the most abundant TR isoform in rodent liver. Here, we have tested if TRalpha1, when expressed at increased levels from its normal locus, can replace TRbeta in regulation of cholesterol metabolism. By the use of TRalpha2-/-beta-/- animals that overexpress hepatic TRalpha1 6-fold, a near normalization of the total amount of T3 binding receptors was achieved. These mice are similar to TRbeta-/- and TRalpha1-/-beta-/- mice in that they fail to regulate cholesterol 7alpha-hydroxylase expression properly, and that their serum cholesterol levels are unaffected by T3. Thus, hepatic overexpression of TRalpha1 cannot substitute for absence of TRbeta, suggesting that the TRbeta gene has a unique role in T3 regulation of cholesterol metabolism in mice. However, examination of T3 regulation of hepatic target genes revealed that dependence on TRbeta is not general: T3 regulation of type I iodothyronine deiodinase and the low density lipoprotein receptor were partially rescued by TRalpha1 overexpression. These in vivo data show that TRbeta is necessary for the effects of T3 on cholesterol metabolism. That TRalpha1 only in some instances can substitute for TRbeta indicates that T3 regulation of physiological and molecular processes in the liver occurs in an isoform-specific fashion.