This study was initiated to define the dose- and time-dependence of triiodothyronine (T3) action on Na-K-ATPase in single microdissected nephron segments. For this purpose, the activity and the number of catalytic sites of Na-K-ATPase, as determined by the specific binding of 3H-ouabain, were measured following a single injection of T3 to rabbits thyroidectomized since 8-12 days. Triiodothyronine restored both the activity and the number of catalytic sites of Na-K-ATPase in a dose-dependent manner in all nephron segments where the enzyme was decreased following thyroidectomy, i.e., the proximal and the collecting tubule. At a dose of 50 micrograms/kg bw, T3 restored Na-K-ATPase activity and 3H-ouabain binding with the same kinetics. However, the kinetics depended on the nephron segments: in the proximal tubule, Na-K-ATPase stimulation occurred after a 12 h period of latency and was completed within 24 h whereas in the collecting tubule, the stimulation was biphasic with a first increase within the first 3 h and a second increase concomitantly to that observed in the proximal tubule. These results indicate that thyroid hormones regulate Na-K-ATPase activity by altering the number of catalytic sites of the enzyme. This control depends on two different mechanisms which differ by their time-dependence.