A radioimmunoassay for quantitatively measuring the serum concentration of free thyroxine is described. This method does not require equilibrium dialysis, and is rapid and reproducible. The serum values obtained by this radioimmunoassay and by equilibrium dialysis are similar in normal subjects, hyperthyroid and hypothyroid patients, pregnant women, "sick euthyroid" patients, and euthyroid patients with hereditary TBG abnormalities. The method also provides a total serum thyroxine concentration in the same assay procedure.