[en] We describe a two-step radioimmunoassay procedure for the measurement of free thyroxine (fT₄) in serum using high affinity antibody immobilized on the inner walls of the polystyrene tubes. The thyroid hormones are circulating in the bound and free form and they exist in dynamic equilibrium. The fT₄ concentration in normal subjects ranges from 7 to 21 pg/ml which is only 0.02% of the total thyroxine present. Hence, high affinity antibody was used to achieve the required sensitivity and the use of extremely small amount of antibody ensured that the bound free equilibrium is not disturbed. In the optimized assay system T₄ sequestration by the antibody from the normal serum sample was kept below 1%. The detection limit of the assay developed is less than 2 pg/ml. The intra and inter assay variability of the assay at low and high clinical limits were less than 10% and 15% respectively. The system developed was compared with the commercial kit using 45 volunteers samples. The fT₄ concentrations measured using our system was comparable with that obtained using the commercial kit. The correlation coefficient was found to be 0.85. (author)