The binding of estradiol (E2) and testosterone (T) to testosterone-estradiol-binding globulin (TeBG) was studied in vivo at 37 C by three independent methods: equilibrium dialysis, steady state polyacrylamide gel electrophoresis, and TeBG-ligand dissociation kinetics. Equilibrium dialysis was performed at 37 C with the dialysate containing human serum albumin in amounts equivalent to that of the plasma dialysand. Scatchard analysis indicated that under these conditions E2 does not measurably bind to TeBG, while T has a Kd of 3.7 X 10(-10) M. Similarly, Scatchard-type analysis of E2 binding to TeBG in steady state polyacryalmide gel electrophoresis at 37 C revealed no high affinity saturable binding, while dihydrotestosterone was bound with a Kd of 2.7 X 10(-10) M. Examination of the dissociation kinetics of T and E2 ffrom TeBG revealed that the mean (+/-SD) T1/2 of dissociation of T from plasma at 37 C (10.8 +/- 2.4 min) was significantly shortened to 3.5 +/- 0.4 min by saturation of plasma with dihydrotestosterone (P less than 0.01), whereas that of E2 (8.9 +/- 1.4 min) was not changed (9.6 +/- 3.0 min). These data suggest that TeBG is not an important binder of plasma E2 at physiological temperatures and explain the observation that in diseases characterized by high TeBG levels, such as hyperthyroidism and liver disease, the MCR and free E2 levels have generally been normal.