The type III iodothyronine deiodinase (ID-III) catalyzes the inner ring deiodination and, thus, the inactivation of the thyroid hormones T-4 and T-3. ID-III activity in rat brain, rat placenta and embryonic chicken liver is inhibited by the affinity label N-bromoacetyl-T-3 (BrAcT3) with an affinity similar to that of T-3. Reaction of rat brain and placenta microsomes with BrAc[I-125]T-3 resulted in the extensive labeling of a 32 kDa protein (p32). However, p32 was also prominently labeled in fetal rat liver microsomes which have no ID-III activity. Labeling of p32 was not influenced by 100 mu M substrate analogs or inhibitors of ID-III, some of which completely inhibit ID-III activity at 1 mu M. BrAc[I-125]T-3 labeling of embryonic chicken liver microsomes did not reveal p32 or another protein possibly related to ID-III, In contrast to previous suggestions, it is unlikely that p32 represents ID-III or a subunit thereof.