The effects of thyroid hormone on Na,K-ATPase alpha-subunit mRNA (mRNA alpha) content and Na,K-ATPase activity were measured in renal cortex, heart, and cerebrum of hypothyroid rats 24 and 72 h after injection of diluent or T3. Use of a cDNA probe complementary to rat brain mRNA alpha in Northern blot analysis revealed a single 26-27 S band in RNA isolated from these three tissues regardless of thyroid status. Tissue mRNA alpha content was estimated by dot blot analysis of whole cell extracts and isolated total RNA. Injection of T3 augmented mRNA alpha content by 2.1- to 2.5-fold in kidney cortex and myocardium at 24 h. After three daily injections of T3, the increases in mRNA alpha were evident despite a global increase in RNA content associated with hypertrophy of these target tissues. Furthermore, the increases in abundance of mRNA alpha after 72 h of T3 treatment correlated with enhancement of Na,K-ATPase activity. In contrast, both mRNA alpha and enzyme activity were invariant in the cerebrum. These data suggest that T3-induced augmentation of Na,K-ATPase activity is mediated, at least in part, by increased mRNA alpha content in target tissues.