The CRH test may sometimes be useful in the differential diagnosis of Cushing's syndrome, because most patients with pituitary ACTH-dependent Cushing's syndrome (Cushing's disease) respond to CRH, but those with other causes of Cushing's syndrome usually do not. However, about 10% of Cushing's disease patients fail to respond to CRH. We wondered if we could eliminate these false negative results either by exploiting the potential additive or synergistic effects of another ACTH secretagogue or by reducing glucocorticoid inhibition of CRH's ACTH-releasing effect. We compared the effect on plasma ACTH and cortisol in 51 patients with Cushing's disease of administering ovine CRH (1 microgram/kg BW, i.v.) alone, arginine vasopressin (AVP; 10 U, i.m.) alone, the combination of CRH and AVP, and CRH after pretreatment with metyrapone (1 g, orally, every 4 h for three doses; CRH + MET). The rates of nonresponse (ACTH increment, < 35%; cortisol increment, < 20%) to AVP and CRH alone were 26% and 8%, respectively; all patients responded to CRH + AVP. The lack of response was not due to improper administration or rapid metabolism of the agonist, because plasma CRH and AVP concentrations were similar in responders and nonresponders. A synergistic ACTH response to CRH + AVP occurred in 65% of the patients. MET pretreatment increased basal plasma ACTH levels in most patients and induced the greatest mean peak ACTH response to CRH, but 8% of the patients did not respond to CRH + MET with an ACTH increment of 35% or more. Because all of the Cushing's disease patients tested in this study responded to the combination of CRH + AVP, whereas 8% failed to respond to CRH alone, we conclude that CRH + AVP administration may provide a more reliable test for the differential diagnosis of ACTH-dependent Cushing's syndrome than administration of CRH alone. Whether this improved sensitivity is accompanied by unaltered specificity for Cushing's disease must be tested in patients with chronic ectopic ACTH syndrome.