A method for the simultaneous measurement of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) and 18-hydroxycorticosterone (18-OH-B) in human peripheral plasma has been developed. The present method consists of extracting plasma with dichloromethane, separating the 18-OH-DOC and 18-OH-B from other steroids on a Sephadex LH-20 column and quantitating each steroid by radioimmunoassay. The mean plasma level of 18-OH-DOC at 8:00 a.m. was 8.2 +/- 3.9 ng/100 ml (mean +/- S.D.) in normal males. It was 7.8 +/- 2.6 ng/100 ml in the follicular phase of normal females and 11.5 +/- 2.8 ng/100 ml in the luteal phase. The corresponding level of 18-OH-B in normal males was 10.3 +/- 4.2 ng/100 ml and in the follicular and luteal phases of normal females was 12.4 +/- 4.5 ng/100 ml and 13.8 +/- 4.1 ng/100 ml, respectively. No sex differences nor difference between the phases of the menstrual cycle was confirmed. Plasma levels of the two steroids were not rarely high in patients with Cushing syndrome due to adrenocortical hyperplasia and carcinoma, primary aldosteronism, idiopathic hyperaldosteronism and congenital 17 alpha-hydroxylase deficiency, while they were usually within the normal range in cases of Cushing syndrome due to adrenocortical adenoma. These steroid levels were significantly low in patients with Addison's disease.