Thyroid hormone regulates the transcription of several important cardiac genes, both structural and regulatory. While the thyroid gland produces predominantly thyroxine (T₄), it is triiodothyronine (T₃ ) which is transported across the sarcolemma, binds to nuclear receptor proteins (TRs) and mediates transcription in the cardiac myocyte. Clinical studies suggest that serum T₃ levels do not accurately reflect cellular T₃ action and the relationships between serum T₃, cellular T₃, TR saturation and the regulation of cardiac specific genes are not completely understood. To address this question, we have studied the dose response relationship of T₃ administered by constant infusion in hypothyroid animals with the simultaneous in vivo transcription rate of the cardiac-specific -MHC gene, measured by quantitating the -MHC heteronuclear RNA (hnRNA) content. Constant infusion of 4 µg T₃/kg body weight/day for 3 days normalized serum T₃ and restored transcription to euthyroid levels. In contrast, daily injections of the same dose failed to maintain serum T₃ at euthyroid levels and -MHC transcription was 55% of that obtained by infusion. While infusion of T₃ at 1.25 µg T₃/kg body weight/day was not sufficient to restore serum T₃ to normal, it was capable of restoring transcription to normal at 3 days, but when administered for 12 days, transcription of -MHC was found to be 50% of euthyroid levels, demonstrating a decreased sensitivity to T₃ over time. Treatment with trichostatin A to inhibit histone deacetylation increased levels of total nuclear acetylated histone H4 by almost 50%, but was without effect on the real time PCR measures of -MHC hnRNA. TSA administered together with T₃ (10 µg T₃/kg body weight) significantly increased transcription of -MHC after 30 hours, thus demonstrating a potential role for histones as cofactors in the T₃ regulation of cardiac -MHC transcription.