Brown adipose tissue (BAT) of hypothyroid rats contains a low Km (type II) iodothyronine 5'-deiodinase (I-5'D) that has been characterized as being insensitive to inhibition by propylthiouracil (PTU), based mainly on observations with homogenates prepared in a medium containing 10 mM dithiothreitol (DTT) and enzymatic assays in the presence of 20 mM DTT in vitro. In the studies reported herein, BAT homogenates from hypothyroid rats prepared in a DTT-free medium were found to contain I-5'D activity at 20 mM DTT, comparable to that in homogenates prepared in a DTT-containing medium, and were activated by submillimolar concentrations of DTT with an EC50 of approximately 0.5 mM. Almost all of the homogenate activities could be accounted for in microsomal preparations. The activity was substantially inhibited by 1 mM PTU. The PTU inhibition was progressively alleviated with increasing concentrations of added DTT and was not seen at DTT concentrations higher than 10 mM. At 250 microM DTT, the Km and maximum velocity values for rT3 and T4 were 2.9 and 1 nM and 70 and 200 fmol/mg protein X h, respectively, with a Ki for PTU of approximately 200 microM. On administration of PTU in vivo (2 mg/100 g BW; 1 h before killing) and subsequent assay at 250 microM DTT, the I-5'D in the homogenates was about 50% inhibited, and the microsomes showed a state of persistent inhibition, with activity levels about 70% of the control value. The data show that BAT type II I-5'D can be substantially activated at submillimolar concentrations of DTT, and this activation is sensitive to inhibition by PTU administered both in vitro and in vivo.