Noniatrogenic Cushing’s syndrome is a rare clinical entity that is caused by an inappropriately elevated endogenous cortisol production and results in significant morbidity and mortality in affected patients. Despite its rarity, Cushing’s syndrome represents one of the most challenging diseases in clinical endocrinology because, despite the use of a variety of tests, its diagnosis and/or differential diagnosis often remains uncertain (1). It has long been known that vasopressin contributes to the regulation of the hypothalamo-pituitary-adrenal (HPA) axis: although it is a weak ACTH-releasing secretagog, vasopressin has a significant synergistic effect with CRH that grossly potentiates pituitary ACTH secretion (2). This action is thought to occur via the specific corticotroph vasopressin receptor also known as V3 (or Vlb) receptor (3). In the past, several investigators have used the administration of vasopressin analog (LVP or AVP) to differentiate between the ACTH-dependent forms of Cushing’s syndrome (4). Thus, positive ACTH and cortisol responses to vasopressin analogs have been observed in the majority of patients with pituitary-dependent Cushing’s syndrome, whereas no such vasopressin tests had a lower diagnostic accuracy compared to the more widely used CRH test (5). Moreover, because of its smooth-muscle constricting actions, administration of vasopressin analogs is commonly associated with several side effects. For these reasons, these tests have not gained wide acceptance for their use in the investigation of patients with Cushing’ s syndrome. Desmopressin, a long-acting analog of vasopressin, appears to be free of the V1 receptor-mediated pressor side effects, and a desmopressin test has been recently introduced in clinical practice as an adjunctive tool in the diagnosis and differential diagnosis of Cushing’ s syndrome (6). In the present chapter, we will review current knowledge on the diagnostic value of this test in the work-up of patients with proven or suspected Cushing’ s syndrome.