Properties of an inactivating pathway of T4 to 3,3'-diiodothyronine (T2') in rat liver homogenate are described. The intermediate product (of this reaction sequence) is reverse T3 (rT3) which is very labile and cannot be measured at pH 7.5. The apparent KM of the reaction rT3 to T2' is 2 X 10(-8) M. The activities catalyzing the reactions T4 to T3, T4 to T2' and rT3 to T2' are found in the 100 000 X g pellet. Propylthiouracil inhibits all three activities to a similar degree; alpha-methyl-p-tyrosine has no effect. T3 seems to be only a minor source of T2' production. The molar ratio of the two T4 deiodination pathways T4 to T3/T4 to T2' under these conditions is about 1.7; however, by changing the pH this ratio could be significantly altered.