We measured cortisol and precursor steroid production in response to ACTH, cholera toxin, and forskolin by the dispersed adrenocortical cells prepared from the adrenal glands of 10 patients with different forms of Cushing's syndrome. The cells prepared from the hyperplastic adrenal glands from 4 patients with Cushing's disease responded in a dose-dependent manner to ACTH, cholera toxin, and forskolin. The adrenal cells prepared from 4 encapsulated adrenal adenomas showed no (n = 2), a lowered (n = 1), or a clear (n = 1) response of cortisol release to ACTH. The cells prepared from the adrenal glands of 1 patient with dysplastic micronodular adrenal glands showed a limited response to ACTH, while the cells from an adrenocortical carcinoma, which secreted very little cortisol per cell, were unresponsive to ACTH, cholera toxin, and forskolin. The reaction of the dispersed adrenal cells from these 10 patients to ACTH, cholera toxin, and forskolin showed a close correlation (P less than 0.001 in all instances). This suggests that the defect in autonomous glands is not located at the level of the ACTH receptor, but, rather, involves the adenylate cyclase complex as a whole or its coupling to cAMP-dependent protein kinase. The release into the medium of the cortisol precursors deoxycortisol, 17-hydroxyprogesterone, and progesterone showed that the four autonomous nodules were characterized by a significantly higher deoxycortisol/cortisol ratio in the medium (P less than 0.01), suggesting a relative blockade of 11 beta-hydroxylase in these adrenal adenomas. This was further substantiated in cells from several adrenals by a significant increase in the release of these precursors in response to ACTH in the absence of a cortisol response. We conclude the following. 1) Adrenal adenoma formation in patients with Cushing's syndrome is accompanied by a parallel decrease in the stimulation of the release of steroid hormones in response to ACTH, cholera toxin, and forskolin. This points to a defect in the adenoma cells beyond the ACTH receptor. 2) Adrenal adenoma formation in patients with Cushing's syndrome is accompanied by a relative blockade of 11 beta-hydroxylase activity. 3) By comparing the preoperative dynamic tests of the pituitary-adrenal axis, the plasma ACTH concentration, the morphology of the adrenal glands, and their in vitro responsiveness, a gradual transition from pituitary to (partial) adrenal autonomy could be recognized in several patients.