Pretreatment of 24 hr.-hypophysectomized rats with intravenous injection of 10 mU of ACTH, 2 hr. before testing, was found to increase the adrenal response to subsequent administration of test doses of ACTH. This result served to develop a modification of the ACTH assay method of Lipscomb and Nelson. Preliminary evaluation suggests that the modification gives good sensitivity (minimum effective dose, 0.02 mU), precision (\~0.2) and a linear dose-response relationship with doses ranging from 0.02 to 0.16 mU of ACTH. Arginine-vasopressin has no influence either given alone or in combination with ACTH. Pharmacological, but not physiological, doses of angiotensin II interfere to some extent with ACTH assay. Blood ACTH levels in rats obtained by the modified Lipscomb-Nelson method agree with those measured by the adrenal ascorbic acid depletion method. Possible mechanisms involved in the effect of ACTH pretreatment on adrenal responsiveness are discussed.