The identity of a dithiol (designated DFB) of relative mass (Mr) = 13,000, reported previously to be present in fraction B of rat liver cytosol and to participate as a cofactor in the 5'-deiodination of iodothyronines, has been investigated. Substitution of highly purified thioredoxin from Escherichia coli for fraction B or of highly purified thioredoxin reductase from either E. coli or rat liver for cytosolic fraction A (containing DFB reductase) permits deiodination of 3,3',5'-[125I]triiodothyronine by rat liver microsomes to proceed. Addition of antibodies to highly purified rat-liver thioredoxin or thioredoxin reductase inhibits deiodination. Thus, the thioredoxin system largely accounts for the activity of the cytosolic cofactor system supporting 5'-deiodination of 3,3',5'-triiodothyronine in rat liver.