A 2.5-fold stimulation of cyclic AMP cellular content is measured 60 sec after addition of 100 nM synthetic ovine corticotropin-releasing factor (C-RF; corticoliberin) to rat anterior pituitary cells in culture. A maximal response of cyclic AMP content at 400% above control is observed between 2 and 30 min after addition of the peptide, whereas an 8-fold stimulation of cyclic AMP released into the incubation medium is measured between 10 and 180 min. A linear 7-fold increase of corticotropin release is observed for up to 3 hr. Preincubation from 18 hr with the potent glucocorticoid dexamethasone has no effect on C-RF-induced cyclic AMP accumulation. The same treatment with dexamethasone causes an 80% inhibition of corticotropin release induced by both C-RF and the cyclic AMP derivative 8-bromoadenosine 3',5'-cyclic monophosphate. The present data show that ovine C-RF is a potent stimulator of cyclic AMP accumulation in rat anterior pituitary cells and that the process is insensitive to the action of dexamethasone. The marked inhibition by dexamethasone of corticotropin secretion induced by a cyclic AMP derivative indicates that glucocorticoids exert their potent inhibitory effects on corticotropin secretion at a step distant to cyclic AMP formation.