Abstract The effects of several substances used for thyroxine-binding globulin blocking in the radioimmunoassay for tri-iodothyronine are described. It was found that anilino-naphthalene-sulfonic acid is the most effective inhibitor of the binding of [ 125I]T 3 to serum proteins. However this substance decreases also the specific binding of the labelled hormone to the antibody. This effect could partially be avoided by the addition of plasma. The same behaviour was found for sodium salicylate, however in this case the initial binding to the antibody could be completely restored by the addition of increasing amounts of plasma. Due to these rather obscure interactions of the labelled hormone with serum proteins and the antibody using these substances a system with high pH buffer and the addition of merthiolate which inhibits also the TBG effects is recommended. There is no influence on antibody binding. Diphenylhydantoin is not useful since the substance is not stable in solution. Tetrachlorothyronine cross-reacts strongly with three different antibodies using two different synthetic lots. Therefore the substance cannot be used in the assay system. Preliminary results showed that using different assay systems the same plasma gives different values for tri-iodothyronine.