A double isotope derivative dilution procedure has been developed to measure deoxycorticosterone (DOC) in human peripheral plasma. The method involves the addition of [14C]-DOC as internal recovery indicator and [3H]-acetic anhydride for derivative formation. The method shows adequate specificity, precision and accuracy. Simultaneously, aldosterone, corticosterone, cortisol and 11-deoxycortisol have been measured with DOC. Normal plasma DOC levels were found to be 6.3 ± 1.0 ng/100 ml (mean ± se, n = 17). The corresponding levels of aldosterone, corticosterone, cortisol and 11-deoxy cortisol were 6.5 ± 1.0 ng/100 ml, 0.42 ± 0.09 μg/100 ml, 13.9 ± 1.4 μg/100 ml and 0.060 ± 0.013 μg/100 ml respectively. ACTH administered to 6 subjects produced a mean increase in plasma DOC from 4.7 to 32.4 ng/100 ml. Angiotensin II infused at subpressor doses for 2 hr into 8 subjects did not increase mean plasma DOC. Similarly, dietary sodium restriction for 5 days or postural change did not increase plasma DOC. These results confirm that DOC secretion is primarily under anterior pituitary control. Eleven patients with Cushing's syndrome were studied. DOC values were elevated in 10 of the patients. Fourteen patients with benign essential hypertension showed a mean plasma DOC level not significantly different from normal. Six out of 10 patients with primary aldosteronism had elevated DOC values. From the basal level of 6.3 ng/100 ml and from its biological activity compared to aldosterone, the major mineralocorticoid, it would seem that DOC plays a minor role in electrolyte homeostasis in normal man. However, the elevated levels seen in some cases of adrenal corticol hyperfunction may mean that DOC contributes significantly to the characteristic symptoms of hypertension and hypokalemia.