Thioredoxin (Thd) and NADPH-Thd reductase, purified to near homogeneity from rat liver cytosol, stimulated, in the presence of NADPH, the 5′-monodeiodination of rT₃ by renal and hepatic microsomes at nanomolar, but not micromolar, substrate concentrations. T₄ was not deiodinated at either concentration. Reduced Thd was effective at physiological concentrations in stimulating microsomal rT₃ deiodination (EC₅₀, -15 μM); Thd-supported microsomal deiodination showed a maximum velocity approximately one third that in the presence of dithiothreitol (DTT), and Thd-supported deiodination, compared to that with DTT, was 10- and 2000-fold more sensitive to inhibition by propylthiouracil and iopanoate, respectively, than was the DTT-supported reaction. The Michaelis constants for rT₃ (2.5 nM) were identical for the Thd- and DTT-activated reactions, suggesting that these thiols stimulated deiodination by the same enzyme. Arrhenius plots also revealed comparable activation energies for Thd- and DTT-mediated low K_m rT₃ monodeiodination; these activation energies were, moreover, distinct from those observed with the low K_m T₄ deiodination in the presence of DTT. The data suggest that renal and hepatic microsomes contain separate low K_m rT₃-specific and T₄-specific 5′-monodeiodinases and that the rT₃-specific monodeiodinase can use the reducing potential of NADPH, via the Thd system. Such an enzyme could mediate the disposal of rT₃, the noncalorigenic metabolite of T₄, independently of the conversion of T₄ to T₃. (Endocrinology121: 1937–1945, 1987)