T he relative roles of the types 1 and 2 iodothyronine deiodinases (D1 and D2) in extrathyroidal 3,5,3′-triiodothyronine (T₃) production in humans are unknown. We calculated the rate of thyroxine (T₄) to T₃ conversion by intact cells transiently expressing D1 or D2 at low (2 pM), normal (20 pM), and high (200 pM) free T₄ concentrations. Deiodinase activities were then assayed in cell sonicates. The ratio of T₃ production in cell sonicates (catalytic efficiency) was multiplied by the tissue activities reported in human liver (D1) and skeletal muscle (D2). From these calculations, we predict that in euthyroid humans, D2-generated T₃ is 29 nmol/d, while that of D1-generated T₃ is 15 nmol/d, from these major deiodinase-expressing tissues. The total estimated extrathyroidal T₃ production, 44 nmol/d, is in close agreement with the 40 nmol T₃/d based on previous kinetic studies. D2-generated T₃ production accounts for approximately 71% of the peripheral T₃ production in hypothyroidism, but D1 for approximately 67% in thyrotoxic patients. We also show that the intracellular D2-generated T₃ has a greater effect on T₃-dependent gene transcription than that from D1, which indicates that generation of nuclear T₃ is an intrinsic property of the D2 protein. We suggest that impairment of D2-generated T₃ is the major cause of the reduced T₃ production in the euthyroid sick syndrome.