The effect of tri-iodothyronine (T3) treatment on myocardial levels of alpha and beta myosin heavy chain (MHC) mRNAs in the rat was defined in vivo and in vitro. Dose-response experiments were performed in intact hypothyroid and euthyroid rats; in addition, studies in vitro examined the effect of T3 on MHC mRNAs in neonatal cardiac myocytes in primary culture. Specific alpha and beta MHC mRNAs were determined by Northern blot and dot hybridization to oligonucleotide probes complementary to the 3' untranslated regions of the MHC genes. An increase in myocardial beta MHC mRNA was demonstrated in hypothyroidism, accompanied by a reduction in alpha MHC mRNA. Marked differences in the sensitivity of alpha and beta MHC mRNAs to T3 replacement were found; a dose-dependent increase in alpha mRNA was evident at 6 h after T3 treatment, in the absence of consistent effects on beta mRNA, whereas 72 h after T3 replacement was commenced, stimulatory effects of T3 on alpha MHC mRNA, evident at all doses, were accompanied by a dose-dependent inhibition of beta MHC mRNA. No effect of thyroid status on actin mRNA was found, indicating the specificity of MHC gene regulation. T3 treatment of cardiac myocytes in vitro exerted similar actions on MHC mRNAs to those found in vivo, with a more marked influence on alpha than beta MHC mRNA. These studies of the action of T3 in vivo and in vitro have thus demonstrated specific effects of T3 on pretranslational regulation of the alpha and beta MHC genes, influences which differ not only in terms of stimulation or inhibition, but also in magnitude of effect.