Synthesis and activity of the enzymatic equivalent of the sodium pump, Na,K-ATPase, are regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine whether triiodothyronine (T3) regulates the level of the messenger RNA (mRNA) coding for Na,K-ATPase α-and β-subunits in the heart. The expression of Na,K-ATPase mRNAs in in vitro myocardial cells was directly assayed by Northern and slot blot hybridization using Na,K-ATPase α- and β-isoform-specific cDNA probes. Exposure of cultured neonatal rat cardiocytes to 10-8M T3resulted in 1) threefold to fourfold increase in α1- and β1-mRNA accumulation, with a maximum elevation at 48 hours, 2) sevenfold increase in α2-mRNA accumulation with a peak elevation at 72 hours, and 3) transient threefold increase in α3-mRNA within the first 24 hours followed by a deinduction thereafter. The increase in αl-mRNA accumulation by T3occurred over the physiological T3concentration range with an EC50of 5×10−10M. This was associated with a twofold increase in αl-subunit protein accumulation and an increase in Na,K-ATPase transport activity. The half-life of α1-mRNA analyzed by actinomycin D chase was less than 3 hours and was not affected by T3. Transfection experiments with the luciferase reporter gene revealed that thyroid hormone response sequences are located within the 5'-flanking regions of each α-isoform gene. The above results suggest that thyroid hormone regulates all three Na,K-ATPase α-isoforms in cardiocytes and may play an important role in the developmental switching of the cardiac α2- and α3-isoforms. These effects are mediated, at least in part, by transcriptional regulatory factors interacting with the respective α-isoform gene promoters.