Context The substitution of liothyronine (L-T3) for levothyroxine (L-T4) is commonly employed during thyroid hormone (TH) withdrawal in preparation for diagnostic and therapeutic interventions on thyroid cancer patients. Presently, only limited data are available on the L-T3 for L-T4 therapeutic substitution. Objective To characterize the pharmcodynamic equivalence of L-T3 and L-T4. Design Randomized, double-blind, cross-over intervention study. Setting NIH clinical center. Patients Ten thyroidectomized patients. Interventions Study participants were treated with L-T3 or L-T4 with a target TSH ≥ 0·5 ≤ 1·5 mU/l for at least 30 days before undergoing inpatient testing. Following testing, subjects crossed-over according to the same scheme. Main outcome measures Area under the serum concentration-time curve of TSH from 0 to 60 min (AUC0-60) and peak TSH serum concentration (Cmax) following thyrotropin-releasing hormone (TRH) stimulation test, total L-T4 and L-T3 dose (mcg/kg), and L-T4/L-T3 ratio. Results No difference was observed for time 0 TSH values between L-T3 and L-T4 replacement phases (1·48 ± 0·77 vs. 1·21 ± 0·62 mU/l, P = 0·293) at average daily doses of 40·3 ± 11·3 mcg L-T3 and 115·2 ± 38·5 mcg L-T4, L-T3: L-T4 ratio 0·36 ± 0·06. TRH stimulation test resulted in similar L-T3 vs. L-T4 TSH responses with AUC0-60 of 326·1 (95% CI 232·6- 457·1) and 247·1 (95% CI 153·8-397·1) mU* min/l (P = 0·285); and Cmax of 6·83 (95% CI 4·88-9·55) and 5·23 (95% CI 3·31-8·3) mU/l (P = 0·383). Conclusions This is the first study addressing the equivalency between L-T3 and L-T4 therapy measured by baseline and TRH-stimulated TSH. The therapeutic substitution of L-T3 for L-T4 was achieved at approximately 1:3 ratio.