Gel filtration of an extract of rat median eminence tissue in 0.1 N HCl on Sephadex G-25 separated two peaks, both with feeble ACTH-releasing activity. Full activity of the extract was regained when both peaks were recombined. This observation suggests that the ACTH-releasing hormone of the hypothalamus requires a co-factor for activity.

The CRF-like activity of peptides related to vasopressin and oxytocin was estimated using a semi-automated in vitro bioassay. Rat pituitary halves were incubated for 1 h with the test material in 1.0 ml of Krebs-Ringer bicarbonate buffer. The ACTH released from the pituitary tissue was estimated using adrenal quarters in an automated superfusion system. The pituitary tissue was standardized with median eminence extract, and adrenal tissue was standardizeed with ACTH. Vasopressin and its analogues release ACTH in order of their pressor potency. Most of the ring or tail fragments of oxytocin and vasopressin did not stimulate the release of ACTH. In some experiments, deaminopressinamide and pressinoic acid had CRF-like activity.

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