A direct radioimmunoassay for the determination of ACTH in a small volume of serum was developed using a commercially available antiserum and tracer. A single-stage radioimmunoassay with an incubation period of 4 days gave reliable and reproducible ACTH values. In the incubation medium 0.1% 2-mercaptoethanol was used as the antioxidant. During a 15-month period the assay has been used routinely in the laboratory. The coefficient of variation was less than 10% in the physiological and pathological range. A normal range of 12 to 60 ng/L was found. The reliability of the assay is demonstrated by results obtained in patients with disorders of the pituitary-adrenal axis.