Abstract Di-iodotyrosine binding to serum protein cannot be demonstrated either by electrophoresis on paper, cellulose acetate or acrylamide-agarose (using various buffers at different pHs) or by double diffusion in agar and immunoelectrophoresis. To the contrary, binding of DIT to serum protein can be estimated by Sephadex G-25 gel filtration and by adsorption on charcoal. Binding increases with pH between 6 and 10. It is almost negligible below pH 6. This property was used to propose a method of separation of all the known iodocompounds found in serum using a single serum sample and two consecutive filtrations on Sephadex G-25. The first filtration at pH 5.80 allows the separation of MIT from DIT and iodide and from PBI. The latter is resolved into T 4, T 3 and iodoprotein by refiltration in 0.01 N NaOH. Recoveries of the radioactive iodine-labeled iodocompounds are almost quantitative. Sephadex gel filtration is useful in estimating the iodotyrosine content of serum. Experiments on DIT clearance from the blood in man support the idea that iodotyrosines are not normal components of the blood of euthyroid humans. The usefulness of Sephadex G-25 gel filtration in 0.01 N NaOH for the quantitative estimation of serum total hormonal iodine is confirmed.