A sensitive and accurate plasma ACTH radioimmunoassay based on that of Yalow and Berson has been developed. To increase the sensitivity of the method 5 ml volumes of plasma were extracted with acid acetone and the extracts were purified on a Sephadex column. The mean plasma ACTH concentration in 10 normal subjects at 8 AM was 0.5 mU/100 ml (range 0.3-0.7 mU/100 ml). A diurnal variation In plasma ACTH levels with maxima at 4 and 6 AM was observed in 2 normal subjects. Plasma corticosteroid levels paralleled the plasma ACTH concentrations. Dexamethasone administration suppressed the diurnal variation in both ACTH and corticosteroid levels in one other normal subject. Patients with panhypopituitarism and normal subjects given dexamethasone had 8 AM plasma ACTH concentrations of less than 0.15 mU/100 ml. Untreated patients with adrenal insufficiency or congenital adrenal hyperplasia and patients with Cushing''s syndrome usually had increased plasma ACTH concentrations.