Various TSH RIA kit components were prepared. Conditions for 125I labelling of h-TSH were optimized by diminishing the amount of chloramine-T, extending reaction time and lowering reaction temperature. Yield, specific activity, and immunological activity could be maintained moderately under such mild reaction conditions. The mixture of polyethyleneglycol (PEG) and second antibody worked effectively as a B/F separation agent. Even though the mixture was made with more diluted PEG and second antibody than those of using the sole component separately, the time required for the B/F separation was shorter in case of using the mixture. The sequential saturation technique was efficient than those of applying ordinary equilibrium saturation technique in assay sensitivity and assay precision points of view.