We have demonstrated that in patients given a single iv injection of T3, rT3, or, to a lesser extent, T4, all labeled with 125I in the outer or phenolic ring, chromatography of serum on columns of Sephadex G-25 superfine revealed the presence of a labeled material, separate from the administered hormone and from both iodide and iodoprotein. This peak has been termed pre-T3 because it elutes just before the T3 peak. Identification of the various compounds in pre-T3 was carried out by cation exchange chromatography. Pre-T3 generated from [125I]T3 consistently contained labeled compounds with the chromatographic behavior of 3,3'-diiodothyronine and 3'-monoiodothyronine, while pre-T3 generated from [125I]rT3 contained labeled products with the chromatographic mobility of 3',5'-diiodothyronine, 3,3'-diiodothyronine, and 3'-monoiodothyronine. In addition, pre-T3 also contained the glucuro- and sulfoconjugates of these several labeled products. These studies demonstrate that T3 and rT3 undergo progressive and probably sequential deiodination in the peripheral tissues, resulting in the formation of a variety of diiodothyronines and monoiodothyronine, as well as their glucuro- and sulfoconjugates.