Thyroxine 5'-deiodinase (type II) is present in a microsomal fraction of brown adipose tissue (BAT) of Syrian hamsters. Cold exposure increased specific activity in homogenates after a lag period of approximately 2 h to reach a maximum by 1-3 days. Total activity increased 80 times in cold-acclimated hamsters. During deacclimation, a rapid decrease occurred again after a lag period of approximately 2 h. The increase did not parallel increases in protein or in thermogenic state of mitochondria (mitochondrial GDP binding), which occurred more slowly. Changes in serum 3,5,3'-triiodothyronine concentration paralleled changes in BAT 5'-deiodinase during both acclimation to cold and deacclimation. The cold-induced increase in BAT 5'-deiodinase activity occurred more slowly but was much larger in hamsters than in rats. Eating a palatable diet increased BAT protein content and thermogenic activity in both hamsters and rats but did not alter total 5'-deiodinase activity in either species. We conclude that the trophic response of brown adipose tissue to cold in both hamsters and rats is accompanied by an enhanced endogenous production of triiodothyronine that may play a role in control of tissue growth. The trophic response to a palatable diet differs in that enhanced endogenous production of triiodothyronine is not an obligatory accompaniment. Specific stimulation of 5'-deiodinase by cold might modify thermogenesis in tissues other than BAT by providing triiodothyronine and be of greater significance in hamsters than in rats.