T₄ must be activated by its monodeiodination to T₃ by type 1 or 2 iodothyronine deiodinase (D1 and D2). Recent studies show that despite an approximately 2000-fold higher Michaelis constant (K_m; T₄) for D1 than for D2 using dithiothreitol (DTT) as cofactor, D1 expressed in intact cells produces T₃ at free T₄ concentrations many orders of magnitude below its K_m. To understand the factors regulating D1 and D2 catalysis in vivo, we studied a mutant D2 with a proline at position 135 of the active center of D2 replaced with a serine, as found in D1. The P135S D2 enzyme has many D1-like properties, a K_m (T₄) in the micromolar range, ping-pong kinetics with DTT, and sensitivity to 6n-propylthiouracil (PTU) in vitro. Unexpectedly, when the P135S D2 was expressed in HEK-293 cells and exposed to 2–200 pm free T₄, the rate of T₄ to T₃ conversion was identical with D2 and conversion was insensitive to PTU. Using glutathione as a cofactor in vitro resulted in a marked decrease in the K_m (T₄) (as also occurs for D1), it showed sequential kinetics with T₄ and it was sensitive to PTU but was resistant when HEK-293 cytosol was used as a cofactor. Thus, the in vivo catalytic properties of the P135S D2 mutant are more accurately predicted from in vitro studies with weak reducing agents, such as glutathione or endogenous cofactors, than by those with DTT.