Thyroxine (T4) and triiodothyronine (T3)-binding interactions in preparations of rat anterior pituitary gland have been studied. T4 is bound primarily to extranulcear binding sites located in the cytosol fraction of the cell. These sites have a medium affinity for T4: Ka = 2.5 x 10(8) 1/mol and a maximum binding capacity (MBC) of 1.15 pmol/mg tissue (wet weight). Binding of T3 to these sites is minimal. The extent of binding of T4 is influenced by the pH of the system and the temperature of incubation. The relative effectiveness of T4 analogues in displacing bound T4 is tetrac greater than T4 greater than triac greater than D-T4 greater than T3. Similar T4-binding sites are present in other rat tissues, but in all except serum, binding activity is lower than in the pituitary. T4-binding by serum contaminating the pituitary preparations contributed only partially to the total activity observed. Concomitant assessment of T4-binding activity and T4 metabolism in pituitary homogenates prepared at different pH values indicated an inverse relationship between the two processes. The possible role of thyroid hormone binding in cytosol in influencing the intracellular distribution of thyroid hormones is discussed.