Normal and pregnancy plasma contains testosterone binding substances. Binding activity is essentially abolished by heating plasma to 60 C or lowering pH to 3H-testosterone using Sephadex G-200 chromatography, immunoelectrophoresis and plasma electrophoresis on paper, indicate that the high affinity binding substance has characteristics of a beta globulin. Testosterone binding globulin (TBG) activity tested by dextran coated charcoal particle adsorption of free labeled testosterone is increased in pregnancy or estrogen administration and appears slightly more active in normal female than male plasma Studies based upon displacement of 3H-testosterone from pregnancy plasma TBG indicate a high degree of stereospecificity for binding which differs completely from the characteristics of cortisol binding protein. TBG binding optimally requires a C-19, 5α-androstane steroid with an unhindered 17β-ol group and secondarily a 3-ol or 3-one group. 5α-Androstane-3β,17β-diol and 17β-hydroxy-5α-androstan-3-one have greater binding affinities than testosterone for this protein, while in the charcoal adsorption technique estradiol-17β has about ¼ the displacement activity. These studies continue to support the concept that the unbound fraction of testosterone in plasma may be the critical parameter to measure.