The effects of various neurogenic peptides and neurotransmitter substances on the release of ACTH induced by hypothalamic corticotropin releasing factor (HYCRF) were investigated using monolayer cultured anterior pituitary cells. Test substances were given in combination with 0.05-0.1 hypothalamic extract (HE)/ml, because HE evoked a significant ACTH release and a linear dose response relationship was demonstrated sequentially between 0, 0165 HE/ml and 0.5HE/ml. Relatively high doses of lysine-vasopressin showed a slight additive effect onl the release of ACTH induced by 0.1HE/ml. Leu-enkephalin, dopamine, prostaglandin E1 and E2 slightly reduced the release of ACTH induced by HY-CRF, but the inhibitory effect of these substances were not dose-related. Other tested substances including luteinizing hormone releasing hormone, thyrottopin releasing hormone, somatostatin, melanocyte stimulating hormone release inhibiting factor, β-endorphin, neurotensin, substance P, vasoactive intestinal polypeptide, angiotensin II, norepinephrine, serotonin, acetylcholine, histamine and γ-amino butyric acid showed neither agonistic nor antagonistic effect on the release of ACTH induced by HY-CRF. These results indicate that the release of ACTH is controlled specifically by HY-CRF and corticosterone, and modified slightly by some other substances such as vasopressin and prostaglandins, and that the effect of most other neurogenic peptides and neurotransmitter substances is negligible or non-physiological at the pituitary level.