The possibility that augmentation of cardiac Na+-K+-dependent adenosine triphosphatase (Na-K-ATPase) by L-3,5,3'-triiodothyronine (T3) was mediated by early changes in intracellular ion concentrations ([Na+]1, [K+]1) was explored by time-course analysis after a single injection of T3 in thyroid-ablated (131I) rats. At 6 and 16 h after injection, T3 had no significant effect on cardiac [Na+]1, [K+]1, or microsomal Na-K-ATPase activity. At 24 and 48 h, however, T3 elicited proportionate increases in [K+]1 and Na-K-ATPase activity. Thus, no evidence was adduced that the T3-dependent increase in ventricular Na-K-ATPase activity is an adaptive response to prior changes in intracellular ion concentrations. The increase in [K+]1 is attributable to an increase in Na+ pump activity. Administration of T3 to hypothyroid rats had no effect on the transition temperature or the activation energy of ventricular microsomal Na-K-ATPase, as analyzed by an Arrhenius plot. Thus, the lipid microenvironment and the properties of the enzyme may be independent of thyroid status. The latter inference was supported by kinetic analysis, in that T3 had no effect on the Km for ATP or the K1/2's for Na+ and K+. Injection of T3 of the hypothyroid rat, however, significantly increased the Vmax's for ATP, Na+, and K+ of ventricular microsomal Na-K-ATPase. These results are in accord with the inference of thyroidal induction of Na-K-ATPase indistinguishable from those present in the athyroid state.