Alterations in the ionic composition of the medium and different secretagogues have been used in order to study the mechanism of release of ACTH from superfused neurointermediate lobes (NIL) of rat hypophysis. We showed that: (a) a tenfold increase of K+ in the medium caused a reversible and repeatable stimulation of the ACTH release; (b) removal of Ca++ reversibly abolished the stimulating effect of high K+; (c) removal of Ca++ had no effect on the stimulating effect of a hypothalamic extract (HE); (d) in the latter case, a reversible significant weakening was obtained by the addition of EDTA in the Ca++-free medium; (e) dbc-AMP caused a reversible and repeatable stimulation of the ACTH release; (f) comparable results were obtained for anterior lobes (AL) superfused in the same conditions. From these data we can conclude that Ca++ is necessary for the stimulation of the hormonal release and that factors such as K+ or dbc-AMP can mimic the in vitro stimulating effect of a HE. Similarities, which appear in this study, between the modulation of the ACTH release from NIL and AL, led us to support the idea that, in vivo, a release of ACTH from the NIL, may occur in physiological conditions.