A method has been devised for the separation and measurement of cortisol and substance S in plasma. The plasma level of substance S was used as an index of pituitary-ACTH release in human male subjects during infusion of the 11β-hydroxylase inhibitor, Metopirone. After eight hours of Metopirone infusion, the mean plasma level of substance S was 9.1 μg per 100 ml. The failure to attain a higher level may be explained, in part, by the rapid rate of clearance of substance S from the plasma. Subjects pretreated with ACTH for three days and tested 48 hours after the last ACTH administration showed a maintained, decreased endogenous release of pituitary ACTH in response to Metopirone testing. Subjects treated with cortisol for 3 days also showed decreased pituitary- ACTH responsiveness and decreased adrenal responsiveness to exogenous ACTH. It is suggested that since the pituitary-ACTH response is decreased by ACTH therapy, this effect may limit the usefulness of ACTH as a therapeutic adjunct during steroid withdrawal.