We have developed a high-dose dexamethasone suppression test that can be administered overnight with a single 8-mg dose and used the new procedure in the differential diagnosis of 83 patients with Cushing's syndrome. In 76 patients with surgically or pathologically proven cause--60 with Cushing's disease, 7 with the ectopic adrenocorticotrophic hormone syndrome, and 9 with adrenal tumors--suppression of plasma cortisol levels to less than 50% of baseline indicated a diagnosis of Cushing's disease. The test had a sensitivity of 92%, a specificity of 100%, and a diagnostic accuracy of 93%. These values equal or exceed those of the standard 2-day test whether based on suppression of urinary 17-hydroxycorticosteroids or plasma cortisol. We conclude that this overnight, high-dose dexamethasone suppression test is practical and reliable in the differential diagnosis of Cushing's syndrome.