Addition of an extract of hypothalamic median-eminence tissue (HME-CRF) (amount equivalent to 0.08 of a piece of HME tissue) to isolated anterior pituitary cells induced a greater increase in ACTH secretion by cells from adrenalectomized than by cells from intact rats. Secretion was induced with little or no latency and rate of secretion was constant over 45 min. Addition of corticosterone to the incubation medium (0.1 µg/ml) inhibited HME-CRF-induced secretion of ACTH in the case of both types of cells, but the onset of the inhibition was more prompt and the degree of inhibition more profound for cells from intact rats. In a second set of experiments, addition of HMECRF to aliquots of isolated pituitary cells from intact and from adrenalectomized rats was progressively delayed after 100 min in vitro exposure of the cells to corticosterone (0.1 µg/ml). ACTH secretion in response to HME-CRF progessively increased with time postwashout of steroid. Estimates of decay of inhibitory action of the steroid are presented. In a third set of experiments, pituitaries were removed at various times after subcutaneous administration of 1.0 mg of corticosterone to adrenalectomized rats. Isolated cells were prepared from the pituitary tissue and challenged with HME-CRF. As judged by secretory response to HME-CRF, time to peak inhibitory action of corticosterone equalled about 8 hr; return of sensitivity to presteroid treatment levels had occurred by 24 hr. The data have been discussed in terms of a dual mechanism of steroid inhibition (Endocrinology94: 1723, 1974)