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The cytochemical assay for ACTH has been adapted into a method for the detection and determination of potential corticotrophin releasing factors. Of the many putative transmitter substances tested, only the posterior pituitary polypeptides resembled hypothalamic extracts in causing dose-related increases in both pituitary ACTH content and release. Vasotocin was the most active of the compounds studied and, unlike the vasopressins, its dose–response relationships closely resembled those of hypothalamic extracts. The increase in ACTH release induced by hypothalamic extract or vasopressin was reduced by corticosterone, cortisol or progesterone but not by testosterone or oestradiol, but the increase in pituitary ACTH content was not affected by any of these steroids.