Previous determinations of plasma ACTH in stressed and non-stressed adrenalectomized rats produced conflicting results. These discrepancies in the study of ACTH secretion were investigated by use of a more sensitive and direct biological ACTH assay. Female rats were anesthetized with ether, and bilateral adrenalectomies were performed. The animals were sacrificed at various intervals between one hour and six weeks, and plasma ACTH was determined during stressful and non-stressful experiments. Stressed adrenalectomized rats demonstrated an immediate and sustained elevation of plasma ACTH from within a few minutes to several weeks. The increased plasma ACTH concentration doubled after three days and stabilized up to six weeks post-adrenalectomy. Non-stressed adrenalectomized rats had non-detectable to low plasma ACTH concentrations during the first 24 hours post-adrenalectomy, but had gradual increasing concentrations thereafter. However, plasma ACTH concentration in the non-stressed adrenalectomized rat never approximated that observed in the stressed adrenalectomized rats or the stressed normal controls. These results indicate that the rat can respond to stress immediately after adrenalectomy if the stress is sufficient. The different responses of the stressed and non-stressed adrenalectomized rat are consistent with different mechanisms of ACTH secretion for acute stress and chronic glucocorticoid insufficiency.