Although corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) have been extensively characterized as stimulators, and glucocorticoids as inhibitors of ACTH secretion, far less is known about the control of the secretion of ACTH precursors from the anterior pituitary or about the types of corticotrophs involved. The present study was designed to systematically evaluate the actions of stimulatory and inhibitory factors on the secretion of ACTH and ACTH precursors (pro-opiomelanocortin, M(r) 31,000; pro-ACTH, M(r) 22,000) from dissociated ovine anterior pituitary cells. The cells were stimulated for 3 h with CRH (10 nmol/l) and AVP (100 nmol/l), alone or in combination with the synthetic glucocorticoid dexamethasone. In designated wells, cells were treated with dexamethasone, (100 nmol/l), beginning 16-18 h before and continuing through the 3-h secretion experiments in the presence of CRH and AVP. Secretion of ACTH-like peptides from intact cultures was compared with that from cultures which had been pretreated with a cytotoxic CRH conjugate (cytotoxin) to eliminate CRH-target cells specifically. Immunoreactive (ir)-ACTH was measured by radioimmunoassay (RIA); ACTH(1-39) and ACTH precursors were specifically measured by two-site immunoradiometric assays that discriminate between the two. In intact populations of cells, dexamethasone had no effect on basal ACTH(1-39) secretion, but decreased the secretion of ACTH(1-39) in response to CRH or AVP. Pretreatment of cells in the same experiments with cytotoxin (for 18 h, beginning 3.5 days before secretion studies) also had no significant effect on basal ACTH(1-39) secretion, but eliminated the response to CRH and decreased the response to AVP.(ABSTRACT TRUNCATED AT 250 WORDS)