The binding of labelled 3,3'-di-iodo-L-thyronine (3,3'-T2) to isolated rat liver mitochondria has been characterized. Specific binding could be detected only in the inner mitochondrial membrane, not in other mitochondrial subfractions. The composition of the incubation medium influenced the binding capacity, the best combination of high specific binding and low non-specific binding being observed in phosphate buffer, pH 6.4. The specific binding of 3,3'-T2 to mitochondria requires low ionic strength: concentrations of K+ and Na+ higher than 10 mmol/l and 0.1 mmol/l respectively resulted in a decreased binding capacity. The optimal calcium ion concentration was in the range 0.01-1.0 mmol/l. Varying magnesium ion, over the range of concentrations used (0.1-100 mmol/l), had no effect. Both ADP and ATP, at over 1 mmol/l, resulted in an inhibition of the specific binding. Incubation with protease resulted in a decrease in specific binding and an increase in non-specific binding, thus indicating the proteic nature of the binding sites. In addition to the above factors in the local environment the thyroid state of the animal might influence the 3,3'-T2-binding capacity. In fact, the thyroid state of the animal seemed not to have an influence on the affinity constant, but it did affect binding capacity.