2-Thiouracil and a number of its alkyl derivatives are known to inhibit the enzymic 5'-deodination of thyroxine to 3,5,3'-tri-iodothyronine. The structural requirements for inhibition of iodothyronine 5'-deiodinase were investigated by using a washed postmitochondrial particulate fraction of human liver. A series of sulphur-containing derivatives of pyrimidine, pyridine, imidazole, benzene and urea, capable of existing in a thiol form, were incubated at several concentrations with the enzyme preparation in the presence of thyroxine and dithioerythritol (cofactor). The degree of inhibition by the respective compounds of the production of 3,5,3'-tri-iodothyronine was studied in relation to their structural features. The major observations were: (i) a free thiol group is essential; (ii) compounds that do not possess a polar hydrogen atom spatially configured so that it is proximal to the thiol group are poor inhibitors; (iii) aromatic characteristics in the presence of requirements (i) and (ii) lead to the expression of potent inhibitory properties; (iv) modification of potent inhibitors by the introduction of hydrophilic substituents reduces the inhibitory potency.