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The plasma cortisol 1 level is the sum of the cortisol bound to the plasma proteins and that which is unbound. During pregnancy or estrogen treatment the plasma cortisol and 17-hydroxycorticoid level rises several-fold (1)(2)(3)(4)(5)(6)(7)(8). This rise is secondary to an increase in the plasma content of transcortin (6, 9), which is a normal a-globulin component of plasma (10) with high affinity for binding cortisol. Since the subject maintained on long term estrogen therapy does not develop the clinical stigmata of hyperadrenocorticism, it has generally been considered that the bound plasma cortisol is physiologically inactive (7,11) and that the unbound plasma cortisol remains at normal concentration (12) in these subjects.In the present investigation the plasma levels of bound and unbound cortisol were determined in a group of estrogen-treated subjects at different adrenal cortisol production rates and compared to those of a control group. At the beginning of this work, we postulated that if the unbound plasma cortisol level is the only factor which determines the extent of the plasma cortisol effect on cellular functions, then the unbound cortisol level should be the same in both groups of subjects when studied at rest. In addition, if only the unbound cortisol level controls pituitary ACTH inhibition via the negative feedback mechanism, then this level should be the same in both groups at equivalent adrenal cortisol production rates. Our results indicate that the unbound cortisol level cannot be the only factor which determines peripheral physiological activity. Estrogen-treated subjects were found to have relatively elevated levels of plasma unbound cortisol.* Submitted for publication November 27, 1963; accepted January 23, 1964. These studies were supported by U. S. Public Health Service grants AM-06085 and RG-9914.1 Cortisol: 1lp,17a,21-trihydroxy-4-pregnene-3,20-dione. MethodsRadioactive cortisol. Cortisol-1,2-H' (2.08 ,uc per ug)2 was purified on two different paper chromatographic systems. This labeled cortisol did not show significant change in specific activity when added to carrier cortisol and recrystallized from three different solvent systems.Ultrafiltrationi procedure. Details of this procedure have been published (13). Ten ml of heparinized plasma was adjusted to pH 7.4, and 0.04 ,tg of tritiated cortisol was then added. The plasma was incubated at 37.5°C for 0.5 hour while being slowly shaken and was then transferred to a cellophane casing and placed in a Toribara ultrafiltration apparatus (14). The sample was centrifuged initially for 15 minutes at 37.5°C. The 0.2-ml ultrafiltrate formed during this period was removed and discarded and the sample further centrifuged for 45 minutes. During this period 0.55 to 0.65 ml of ultrafiltrate formed. One-half ml of the ultrafiltrate was transferred to a liquid scintillator (15) and counted in a liquid scintillation spectrometer. The number of counts in the 0.5 ml of ultrafiltrate was considered representative of the proportion of unbound cortisol...