A fundamental study of testosterone-estradiol binding globulin (TeBG) activity for estradiol-17beta was investigated by equilibrium dialysis. In addition, the interaction of TeBG activity for estradiol and estradiol metabolism during a menstrual cycle and in hyperthyroidism was attempted to elucidate. Estradiol in plasma and 24 hrs urine was measured by radioimmunoassay. TeBG binding capacity (TBC) was measured by ammonium sulfate method and associate constant (Ka) of TeBG for estradiol was estimated by equilibrium dialysis. 1. In several pooled plasmas, the mean Ka of TeBG for estradoil was 1.25 X 10(8) L/M at 4 degrees C and 0.78 X 10(8) L/M at room temperature. The Ka decreased with increasing temperature and could not be determined with a sufficient accuracy at 37 degrees C. The mean TBC was 4.4 X 10(-8) M/L. 2. TBC and Ka of TeBG for estradiol during the normal menstrual cycle of a woman were fairly unchanged. The unconjugated estradiol value in urine showed the same biphasic pattern with a midcycle peak as that of the unconjugated estradiol value in urine. 3. A normal man was induced hyperthyroidism with a administration of triiodothyronine. The treatment of triiodothyronine caused a remarkable increase of TBC, but no changes of Ka of TeBG for estradiol and unconjugated estradiol values in plasma and urine. From these results it was suggested that the rate of metabolism of estradiol increased in hyperthyroidism.