Studies using thyroid hormone analogs have provided insight into the structural requirements for thyromimetic activity and for thyroid hormone binding to thyroxine-binding globulin, thyroxine-binding prealbumin, and nuclear T3 receptors. To determine the structural specifications for iodothyronine interaction with 5'-iodothyronine deiodinase (5'-ITD), we examined the ability of 35 thyroid hormone analogs to inhibit hepatic T4 5'-deiodination in vitro. The compounds were incubated in concentrations of 0.1-500 microM with rat liver homogenates, and concentrations producing 50% inhibition of T3 production were calculated. Those iodothyronine analogs which likely serve as substrate for 5'-ITD, e.g.rT3 and 3',5'-T2, and those which have one tyrosyl iodide were the most potent inhibitors of 5'-ITD activity. The presence of tyrosyl iodides enhanced inhibition by compounds with alkyl and halogen substitutions. Inhibition was likely due to direct interaction with the enzyme, since it was readily reversed by DTT. The terminal amino and phenolic hydroxyl groups, as well as the ether linkage, do not appear to be essential components of enzyme interaction.