Extracts of rat stalk-median eminences were chromatographed on Sephadex G-50 and BioGel P2. Effluent fractions were monitored for corticotrophin-releasing factor (CRF) bioactivity, using the perfused isolated pituitary cell column bioassay, and radioimmunoassay for CRF-41, AVP, oxytocin, neurophysin, adrenocorticotrophic hormone (ACTH) and luteinizing hormone-releasing hormone (LHRH). Porcine hypothalami were also chromatographed on Sephadex G-50. Our results suggest that vasopressin and a peptide in the rat related to synthetic ovine CRF are two components of the hypothalamic CRF complex which potentiate each other at the corticotrope to release ACTH. However, these two substances alone do not appear to be wholly responsible for CRF bioactivity of hypothalamic stalk-median eminence extracts and other CRFs may yet be found.