It was observed in the present investigation that labeled thyroxine (T4) comprised less than 2% of the total 131I in the thyroids of severely iodine-deficient rats labeled with 131I for 18-24 h, a much lower value than had previously been reported for iodine-deficient rats. This low value was attributable to two factors: 1) the use of a diet low enough in iodine content to produce extreme iodine deficiency, and 2) the use of a paper chromatography system that successfully separates T4 from the minor iodothyronines, 3,3'-diiodothyronine (T2) and 3',5',3-triiodothyronine (reverse T3; T3'). Formation of the minor iodothyronines, while low, becomes appreciable in relation to T4 formation in severe iodine deficiency. In the present study, the formation of labeled T2 was significant only in iodine deficiency, and the highest values were observed in the most severely iodine-deficient rats. In the latter, labeled monoiodotyrosine (MIT) comprised approximately 60% of the total 131I in the thyroid, and the increased formation of T2 could be attributed to the increased probability of coupling between two molecules of MIT. The formation of labeled T3', on the other hand, was significant in thyroids from both iodine-deficient and iodine-sufficient rats. Similarly, in thyroglobulin iodinated in vitro with thyroid peroxidase to varying levels of iodination, the formation of T2 was evident only at lower levels of iodination, whereas the formation of T3' was significant at all levels of iodination. The comparison of relative T3' and T4 formation in enzymatically iodinated thyroglobulin with corresponding values reported for the intermolecular (DIHPPA) model for T4 formation, indicates that the peroxidase model system simulates much more closely the relative formation of T3' and T4 seen in vivo.