Incubation of anterior pituitary (AP) fragments of rats was used to determine the specific role played by vasopressin (VP) in the overall effect of crude hypothalamic median eminence (HME) extract on ACTH release. Using the property of an AVP antiserum (AS) to completely abolish the CRF-like effect of hypothalamic VP without apparently affecting the effect of CRF, we show that under specific incubation conditions, the effect of the two secretagogues are additive at the pituitary level. ACTH secretion of pituitaries was enhanced when incubation was carried out in the presence of VP together with a maximum effective dose of VP-free HME extract (from Brattleboro rats). These observations favor the hypothesis that VP and CRF have different receptor sites in the anterior pituitary.