In 15 untreated patients with Cushing''s disease the regulation of prolactin (PRL) was evaluated. Plasma PRL was 11.5 .+-. 4.8 vs. 5.3 .+-. 3.6 ng/ml (patients with Cushing''s disease vs. controls; mean .+-. S.D.; P < 0.001). The maximal increment of plasma PRL in response to TRH [thyroliberin) was 32.3 .+-. 17.3 vs. 27.9 .+-. 17.2 ng/ml (NS [not significant]; the maximal increment of plasma PRL in response to an insulin-induced hypoglycemia was 3.8 .+-. 4.6 vs. 22.7 .+-. 12.4 ng/ml (P < 0.001). The effect of dexamethasone, lysine vasopressin and ACTH on the secretion of PRL by rat pituitary glands in vitro was also studied. Dexamethasone (1.25-10 .mu.M) inhibited the secretion of PRL. However, in the presence of dexamethasone modulation of PRL release by TRH and dopamine remained unaltered. Lysine vasopressin (5 nM-5 .mu.M) and ACTH (0.5-12.5 .mu.M) did not have a direct effect on PRL release by normal rat pituitary glands in vitro and these substances also did not interfere with dopamine-mediated inhibition of PRL release. In Cushing''s disease the PRL responses to TRH (normal) and to insulin-induced hypoglycemia (blunted) are differentially affected. Hypercortisolism probably selectively interferes with the regulation of PRL secretion at a suprahypophyseal level. TRH and dopamine apparently regulate PRL release at sites which are not under corticosteroid regulation, while corticosteroids modulate PRL secretion in response to stress.