Polyethyleneglycol (PEG) has been recommended as a separating agent in the assay of some peptide hormones (Desbuquois, B. and Aurbach, G.D. (1971) J. Clin. Endocrinol. 33, 732) and several substances of low molecular weight (Ratcliffe, J.G. (1974) Br. Med. Bull. 30, 32). In the present study the PEG-separation technique has been modified and adapted for the assay of thyroid hormones. Separation with PEG has the advantage of being cheap, rapid and relatively non-susceptible to disturbances as compared with the charcoal and double-antibody-solid phase techniques. The influence of different buffer systems, varying pH and ionic strength, on the precipitation process with PEG also has been investigated. Of the different systems tested barbital buffer containing 0.1% human serum albumin proved to be the best, preferably in the presence of bovine gamma-globulin. In the radioimmunoassay of T3 variations in pH and ionic strength are of minor importance whereas in the radioimmunoassay of T4 the adherence to a certain pH is recommended. Barbital buffer containing 0.1% bovine serum albumin was inadequate in the T3 radioimmunoassay, while Tris and phosphate buffers did not give satisfying results for either radioimmunoassay.