Rat anterior pituitary cells were placed in a superfusion column and were stimulated by rat hypothalamic extract. The rate of ACTH secretion was monitored by collecting fractions eluting from the column every 2 min. Dispersed rat adrenocortical cells were used to determine the amounts of ACTH in the superfusate. ACTH appeared in the medium 6-12 sec after stimulation of the cells. A linear log dose-response relationship existed between 1/8 to 1 rat hypothalamus extract and the amounts of ACTH released. Repeated pulses of CRF stimulation of the same column of pituitary cells resulted in repeated, identical peaks of ACTH. Constant stimulation caused a plateau in hormone release that lasted as long as the stimulus was present. The stimulated, but not the basal ACTH release, could be inhibited by superfusion with corticosterone in vitro (0.2 mug/ml), or by treating the pituitary donor animal in vivo with corticosterone (5 mg/100g BW), cortisol or dexamethasone. Adrenalectomy of the donor animal caused increases in the basal and the stimulated ACTH release.