To understand the roles of thyroid hormone receptors (TRs) in adipogenesis, we adopted a loss-of-function approach. We generated 3T3-L1 cells stably expressing either TRalpha1 mutant (TRalpha1PV) or TRbeta1 mutant (TRbeta1PV). TRalpha1PV and TRbeta1PV are dominant negative mutations with a frameshift in the C-terminal amino acids. In control cells, the thyroid hormone, tri-iodothyronine (T(3)), induced a 2.5-fold increase in adipogenesis in 3T3-L1 cells, as demonstrated by increased lipid droplets. This increase was mediated by T(3)-induced expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding protein alpha (C/EBPalpha), which are master regulators of adipogenesis at both the mRNA and protein levels. In 3T3-L1 cells stably expressing TRalpha1PV (L1-alpha1PV cells) or TRbeta1PV (L1-beta1PV cells), adipogenesis was reduced 94 or 54% respectively, indicative of differential inhibitory activity of mutant TR isoforms. Concordantly, the expression of PPARgamma and C/EBPalpha at the mRNA and protein levels was more repressed in L1-alpha1PV cells than in L1-beta1PV cells. In addition, the expression of PPARgamma downstream target genes involved in fatty acid synthesis - the lipoprotein lipase (Lpl) and aP2 involved in adipogenesis - was more inhibited by TRalpha1PV than by TRbeta1PV. Chromatin immunoprecipitation assays showed that TRalpha1PV was more avidly recruited than TRbeta1PV to the promoter to preferentially block the expression of the C/ebpalpha gene. Taken together, these data indicate that impaired adipogenesis by mutant TR is isoform dependent. The finding that induction of adipogenesis is differentially regulated by TR isoforms suggests that TR isoform-specific ligands could be designed for therapeutic intervention for lipid abnormalities.