This study was undertaken to examine the performance of the overnight dexamethasone test, i.e., plasma cortisol level at 8-9 AM following dexamethasone 1 mg by mouth at midnight, in screening for Cushing's syndrome. The participants included 19 patients with Cushing's syndrome (17 with Cushing's disease, 1 with adrenal carcinoma, and 1 with ectopic ACTH syndrome) and 96 patients in whom the possibility of Cushing's syndrome was raised but who did not have the disorder. Utilizing our original very conservative cutoff point of plasma 100 nmol/L (3.62 micrograms/dL) above which Cushing's syndrome was suspected, the sensitivity of the test was 100% but the false positive rate was an unsatisfactory 12.5%. However, the lowest plasma cortisol level achieved following the overnight dexamethasone test in patients with Cushing's syndrome was 259 nmol/L (9.39 micrograms/dL). A plasma cortisol cutoff point of 250 nmol/L (9.06 micrograms/dL) yielded no false negatives and the false positive rate fell to 6.25%. Using a cutoff point of 200 nmol/L (7.24 micrograms/dL) the false positive rate was 7.3%. These performance characteristics of the overnight dexamethasone test compare very favorably with the reported experience of all other screening procedures for Cushing's syndrome including the urinary free cortisol excretion rate and the 48 h dexamethasone test, while the overnight dexamethasone procedure is the simplest for both patients and medical personnel. Since it is possible that some very unusual patients may suppress to plasma cortisol levels lower than that seen in the present study, we now recommend the overnight dexamethasone test using a plasma cortisol cutoff point of 200 nmol/L as the procedure of choice when screening for patients with Cushing's syndrome.