Endogenous corticosteroid hormones and other steroids were removed from sera by gel filtration over Sephadex at 45[degree]. At this temperature, the association constant of the corticosteroid-binding globulins (CBG)-corticosteroid complex is many times smaller, and the rate of dissociation considerably higher than at 4[degree]. The removal of exogenous hormone was practically complete; the CBG activity was not affected. Binding affinity of the filtered sera was determined by equilibrium dialysis in the presence of radio-labeled cortisol, corticosterone, and progesterone at different levels, at 4[degree] and 37[degree]. Concentrations of CBG binding sites and apparent association constants for the various steroid hormone-CBG complexes were derived from the dialysis data. The concentration of binding sites was found to be approximately the same for the three steroid hormones studied, ranging from 3.4 X 10-7 [image] for rabbit serum to 11.3 X 10-7 [image] for rat serum, with an intermediate level of 7.2 X 10-7 [image] for rat serum, with an intermediate level of 7.2 X 10-7 [image] for human serum. The association constant at 4[degree] was between 10 and 40 times greater than at 37[degree]. As far as comparisons can be made, the values presented are in good agreement with those reported from other laboratories.
