The concentration of serum thyroxine (T4)-binding globulin (TBG) not binding T4 (unsaturated TBG, u-TBG) was determined in hyper- and hypothyroidism. u-TBG was expressed as the product of TBG concentration and the ratio of free TBG capacity to maximal TBG capacity as determined by reverse-flow electrophoresis. u-TBG concentration in normal sera (n = 40) was 15.5 +/- 2.3 mg/l (mean +/- SD), or 257 +/- 38 nmol/l for a molecular weight of TBG of 60 000 daltons. u-TBG levels were significantly lower in hyperthyroidism (7.1 +/- 2.3 mg/l, n = 16, P less than 0.001) and higher in hypothyroidism (21.7 +/- 5.0 mg/l, n = 22, P less than 0.001). Based on partial correlation analysis, u-TBG was inversely correlated to serum T4 (r = -0.586, P less than 0.001), but not correlated to triiodothyronine (T3) (r=-0.180, NS). There was a reciprocal correlation between u-TBG concentration and the T3 uptake value (r = 0.748, P less than 0.001). There was also a reciprocal correlation of u-TBG with both % free T4 (r = 0.425, P less than 0.001) and % free T3 (r = 0.377, P less than 0.001), when the data were subjected to partial correlation analysis. These results provide the values for u-TBG concentration in hyper- and hypothyroidism, and support the concept that the free fractions of serum thyroid hormones may be determined by the number of binding sites of the TBG molecule that are not saturated with T4 in hyper-and hypothyroidism.