It is unclear whether Cushing's disease results from a primary pituitary disorder or arises in response to abnormal hypothalamic control of the pituitary gland. Clonal analysis can provide information as to whether neoplastic tissue is derived from a monoclonal proliferation of a genetically altered cell or from a polyclonal expansion of a group of cells affected by a common stimulus. We used X-linked restriction fragment length polymorphisms at the phosphoglycerate kinase, hypoxanthine phosphoribosyltransferase, and DXS255 loci in 11 women with biochemically and pathologically confirmed Cushing's disease to determine the clonal origins of corticotroph adenomas and corticotroph hyperplasia. Tumor tissue from all 10 women with morphologically and immunohistochemically confirmed ACTH-secreting pituitary microadenomas demonstrated a monoclonal pattern. Pathologically confirmed corticotroph hyperplasia in a patient with a CRH-secreting bronchial carcinoid was found to be polyclonal. We conclude that corticotroph microadenomas in Cushing's disease are monoclonal, supporting the theory that a spontaneous somatic mutation is the primary pathogenetic mechanism in this disorder. In addition, the demonstration of polyclonality in corticotroph hyperplasia implies that excess of hypothalamic hormones is an etiologic mechanism in cases of Cushing's syndrome associated with ectopic CRH-secreting tumors.