Cortisol production is appropriately maintained by a complex control system which involves primarily the hypothalamus, the pituitary, and the adrenal cortices. Very small quantities of ACTH stimulate cortisol production, and maximum stimulation occurs with serum concentrations of only 3 mU/100 ML. Under normal circumstances, cortisol is secreted in bursts about ten times each day and circulates predominately bound to a specific binding protein which is rather completely saturated. Radioimmunoassay of plasma cortisol is now generally available and is the assay method of choice, but because of the episodic nature of its secretion, random values of plasma cortisol must be interpreted with great reservation, and even the comparison of morning and evening values in assessing circadian rhythmicity is not often helpful. Urinary free cortisol determinations provide excellent discrimination between normal function and all forms of hypercortisolism. Although the response of the adrenal cortices to ACTH may be evaluated in a number of different ways, the simplest but most definitive procedure involves continuous intravenous administration over a 48-hr period. Of the various tests which indirectly assess the potential for ACTH secretion, the use of metyrapone is most helpful. In the test of greatest utility, plasma cortisol and 11-desoxycortisol are determined the morning after a single midnight oral dose of 30 mg/kg. The detection of all forms of pathologic hypercortisolism is still best accomplished by the oral administration of dexamethasone. Plasma cortisol can be determined the morning after a single midnight dose of 1 mg, or urinary 17-hydroxycorticosteroids can be determined after 2 days in which 0.5 mg is given at 6-hr intervals. Patients with hypercortisolism of hypothalamic-pituitary origin usually evidence appropriate suppression of urinary steroids if the dose is increased to 2.0 mg every 6 hr for another 48 hr. In patients who do not suppress on this or even higher doses of dexamethasone, the distinction between those with adrenal tumor and those with the ectopic ACTH syndrome can be accomplished most definitively by the assay of plasma ACTH where this determination is available.