A radioimmunoassay for human thyroxine-binding globulin (TBG) has been developed. It employs highly purified TBG as an antigen, an antiserum prepared against the TBG, and 125I-labeled TBG. The assay is specific, being unaffected by sera from animals or by human serum proteins other than TBG. It can detect less than 1 ng of TBG. TBG concentration in sera from normal adults was found to range between 2.0 and 4.8 mg/100 ml, with a mean of 3.6. Severe cirrhosis of the liver caused a significant lowering of TBG concentration. Hypothyroidism was without apparent effect. Except in thyroid disease, TBG concentration was found to correlate with the result of the T3 resin sponge uptake test (T3RU); in hyperthyroidism the T3RU is higher than predicted from the TBG concentration and the opposite is true in hypothyroidism. In all sera tested the maximum binding capacity of TBG in whole serum for T4 was found to be proportional to the TBG content. In subjects with congenital disorders of TBG concentration, the result of the TBG immunoassay corresponded with the expected change in TBG concentration based on genetic data and on the binding of T4 to TBG. This included subjects with no detectable binding of T4 to TBG, in none of whom was immunoreactive TBG found.