Using incubated glands, we showed that cerebral cortex and liver extracts (CCE and LE) stimulated ACTH release from neurointermediate lobe (NIL) of hypophysis as well as hypothalamus extract (HE) did. Moreover, the HE-induced ACTH release was much smaller for the NIL (1.9 × basal level) than for the anterior lobe (AL; 13.7 × basal level). Thus, under these conditions, HE seemed to have no specific effect on NIL ACTH release. Using superfused glands, we showed: (a) that both spontaneous and HE-induced ACTH release decreased during superfusion; (b) that using this system, a specific stimulatory effect of HE on NIL was observed. In contrast to HE, CCE and LE had only a small effect on NIL ACTH release (always less than 20% of that caused by HE) which could be considered as a nonspecific response; (c) that trypsin suppressed the stimulating effect of HE as well on NIL as on AL; and (d) that arginine antidiuretic hormone (ADH) was not responsible for the stimulating effect of HE on NIL ACTH release, because synthetic ADH had no effect and HE containing ADH (from normal rats) or HE containing no ADH (from Brattleboro rats or from immunoneutralization of ADH in normal HE) had the same effect. From these results, we can conclude that HE contain a peptidic factor different from ADH which is able to stimulate in vitro release of ACTH from the NIL.