Male and female rats, both intact and castrate, were decapitated and fractions of anterior pituitary homogenates were prepared by centrifugation. Sephadex G–200 gel nitration of incubated 105,000 g cytosol samples showed specific binding of 11.4 and 12.8% of added 3H—estradiol to a similar frontally—eluted macromolecular component in male and female samples, respectively, but no 3H—testosterone binding. Sucrose gradient centrifugation patterns showed specific estradiol binding to form an 8S complex; concentration of 8S—bound estradiol, in pmoles/mg cytosol protein, was 0.37 for male cytosol and 0.36 for female cytosol. 3H—Testosterone did not form a detectable 8S complex. Equilibrium dialysis of cytosol samples showed consistently high and constant uptake of estradiol. Extent of high—affinity interaction was calculated after redialysis and gel nitration as 0.12 pmoles/mg cytosol protein. (Endocrinology92: 322, 1973)