A new procedure for radioimmunoassay (RIA) was applied to determine the serum concentration of thyroxine (T4). Mixtures of T4-methylester hydrochloride-bovine serum albumin complex and complete Freund's adjuvant were injected to rabbits to get them immunized. Specific anti-T4 rabbit serum, high in titer, could be obtained 18 months after the start of immunization. RIA for T4 extracted by ethanol from serum was performed by an application of dextran-coated charcoal to separate bound and free 125-i-t4. on the other hand, RIA for T4 performed directly in the serum containing 100 mg/100 ml of 8-anilino-1-naphthalene-sulfonic acid (ANS) gave a similar standard curve for T4 obtained by the ethanol-extraction method. Serum T4 values obtained by the methanol-extraction method were 9.7 +/- 3.3 mug/100 ml for patients with hyperthyroidism and 0.6 +/- 0.3 mug/100 ml for those with hypothyroidism. The corresponding figures obtained by RIA in the serum containing ANS were 11.1 +/- 2.1, 19.1 +/- 3.3 and 0.7 +/- 0.7 mug/100 ml, respectively. The figures positively correlated respectively to the serum T4 values obtained by the competitive protein-binding analysis method (Tetrasrob-125).