Tissue from histologically confirmed ACTH cell adenomas in Cushing’s disease (CD) and Nelson’s syndrome (NS) was gained by transsphenoidal surgery. Combined enzymatic and mechanic agitation of tumor tissue yielded a cell suspension. Aliquots of the cell suspension were transferred to superfusion chambers immediately after isolation and investigated for ACTH and (3-endorphin production. Feedback action of cortisol (CO) and dexamethasone on basal hormone production and on lysine vasopressin (LVP) induced ACTH secretion were studied. Adenomatous tissue and anterior lobe tissue from the same patient in CD could be investigated simultaneously in 4 cases. The paraadenomatous tissue showed depression of basal and LVP-induced ACTH secretion. In all adenomatous tissues investigated there was missing or reduced suppression of basal ACTH secretion by physiological levels of CO. CO not only failed to suppress LVP-induced ACTH secretion but also seemed to enhance LVP stimulation in some experiments. This study confirms former results, that a missing or inversed feedback action of glucocorticoids in adenoma cells is a mechanism involved in the pathological ACTH secretion in CD and NS. Bioassayable and immunoreactive ACTH from media of superfusion and short-term static incubation were compared with β-endorphin and β-LPH in an assay detecting these two peptides with equimolar sensitivity. Secretory patterns were basically parallel but great differences showed in quantities of hormones secreted. In addition, Sephadex G-50 gel chromatography was performed to separate β-endorphin from β-LPH and to calculate the ratios. These profiles show great variations between different adenomas.