Bacteremia has been associated with pituitary-adrenal activation, both experimentally and clinically. Esch. coli bacteremia was produced in intact 200-g Sprague-Dawley male rats (5) given viable organisms (4.35 × lO8) intravenously. Two hr later plasma ACTH activity was increased more than 3-fold as compared to that of rats given saline. ACTH content of plasma and incubation fluids (below) was assayed in 2-hr hypophysectomized rats, potency being determined by increments in adrenal venous corticosterone secretion rate. In separate in vitro studies of ACTH release, incubations (15) of surviving (untreated) rat anterior pituitary segments liberated 1.24 mU ACTH/60 min. This release rate was not altered during 50 subsequent incubations by addition of Esch. coli (1.17 mU/60 min), a purified Esch. coli endotoxin (1.25), arginine vasopressin (1.19), angiotensin (1.32) or norepinephrine (1.93). Significant augmentation (p under 0.01) of in vitro ACTH release was obtained with only 2 materials, lysine vasopressin in high flask concentrations (0.6–12 μg/ml) releasing 2.21 mU, and crude suspensions of (one) rat hypothalamus, 3.78 mU/60 min. Neither bacteria nor other materials (above) further augmented the releasing action of hypothalamic suspensions, nor did they simulate, destroy or potentiate ACTH. These studies indicate that Esch. coli, while eliciting ACTH release in vivo, has no direct ACTH-releasing action on pituitary tissue in vitro. Physiologic amounts of only one material obtained from single rat hypothalami liberated ACTH directly from rat adenohypophysial tissue in vitro. It is concluded that bacteremia-induced ACTH release requires extrapituitary mediation, possibly via a factor present in hypothalamus. (Endocrinology74: 894, 1964)