We measured thyroxine 5'-deiodinase I (T(4)5'D-I) activity in thyroid, liver, and kidney and thyroxine 5'-deiodinase II (T(4)5'D-II) activity in brown adipose tissue (BAT) in rats on a low-iodine diet (LID) to test the possibility that increased deiodinase activity in these tissues might contribute to the maintenance of ther serum 3,5,3'-triiodothyronine (T3) level. Control rats received LID plus KI. Experiments were also performed with LID and LID plus KI rats exposed to cold. T(4)5'D-I activity was greatly increased in the thyroids of LID rats but not in liver or kidney. We consider it likely that increased thyroxine (T4)-to-T3 conversion in the greatly enlarged thyroids of LID rats contributed to the maintenance of serum T3. T(4)5'D-II activity in BAT was markedly increased in LID rats and was further greatly increased on cold exposure. However, we were unable to demonstrate an increase in uncoupling protein mRNA levels in BAT in response to cold in LID rats. We attribute this to the very low serum T4 level, which limits substrate availability. This factor also makes it unlikely that BAT contributes to maintenance of serum T3 in LID rats.