Abstract: Type I iodothyronine deiodinase (5′-DI) generates the thyromimetically active hormone 3,5,3′-triiodothyronine (T3) by reductive monodeiodination of the phenolic ring of L-thyroxine (T4). The present study was undertaken to compare the effects of cold stress (4°C) alone or in combination with immobilization stress (IMO), insulin treatment (5 IU/kg, i.p.) or 2-deoxy-D-glucose (2DG)-induced intracellular glucopenia on the activity of 5′-DI in rat liver. Cold stress either for 24 h or 28 days when compared to that in the nonstressed group of rats significantly reduced (P < 0.001) the activity of 5′-DI in liver. In comparison with cold stressed rats for 28 days, an additional decrease in 5′-DI activity was observed when those rats underwent 1 × IMO in cold for 2 h (P < 0.001) or insulin treatment (P < 0.05). A significant decrease in 5′-DI activity was found also in rats maintained at room temperature that underwent a single 1 × IMO for 2 h (P < 0.001) or insulin treatment (P < 0.01) when compared to nonstressed animals. In comparison with nonstressed rats, no significant change of the 5′-DI activity was observed after 2DG application (500 mg/kg, i.p.) at room temperature. In conclusion, cold stress and/or 1 × IMO, insulin treatment, or 1 × IMO at room temperature markedly affects reductive monodeiodination of T4, and thus reduces the concentration of biologically active T3 in liver.