Triiodo-L-thyronine (T₃) is essential for maintaining normal cardiac contractile function by regulating transcription of numerous T₃-responsive genes. Both hormone availability and relative amounts of nuclear thyroid hormone receptor isoforms (TR1, TR1) determine T₃ effectiveness. Cultured neonatal rat ventricular myocytes grown in T₃-depleted medium expressed predominantly TR1 protein, but within 4 hours of T₃ treatment, TR1 protein increased significantly, whereas TR1 was decreased by 46 ± 5%. Using replication-defective adenoviruses to overexpress TR1 in cardiomyocytes, we studied the mechanisms by which T₃ mediated the decrease in TR1 protein. Inhibitors of the proteosome pathway resulted in an accumulation of ubiquitylated TR1 in the nucleus, and prevented T₃-induced degradation of ubiquitylated TR1, suggesting that T₃ induced proteosome-mediated degradation of TR1;however, TR ubiquitylation was T₃-independent. TR1 transcriptional activity, measured using transient transfection of a TRE-reporter plasmid, was T₃-dose dependent and inversely proportional to nuclear TR1 content, with 10 nM T₃ having maximum effect. Quantitative RTPCR showed that both endogenous and adenovirus expressed TR1 mRNAs were significantly decreased to 54 ± 11% and 25 ± 5%, respectively, within 4 hrs of T₃ treatment. Measurements of TR1 mRNA half-life in actinomycin D treated cardiomyocytes, showed that T₃ treatment significantly decreased TR1 mRNA half-life from 4 hrs to less than 2 hrs, while it had no effect of TR1 mRNA half-life. These data support a role for both the proteosome degradation pathway and altered mRNA stability in T₃-induced decrease of nuclear TR1 in the cardiomyocyte, and provide novel cellular targets for therapeutic development.