Initially the hypothalamic factor responsible for the release of corticotropin (CRF), was thought to be a simple peptide. More recent work has led to the conclusion that CRF is a multifactorial complex. In 1979 we proposed that vasopressin, much disputed as a CRF candidate, was a major constituent of the complex, interacting with a potentiating the CRF activity of the other component(s). The recent characterization of a 41 residue ovine hypothalamic peptide capable of releasing adrenocorticotropic hormone (ACTH) in a dose-related manner has allowed us to compare its CRF bioactivity with that of vasopressin and simple extracts of the hypothalamus, and to investigate any interaction it may have with vasopressin and other hypothalamic factors in the release of ACTH. We report here that the new CRF is more potent than vasopressin in releasing ACTH. When given simultaneously with vasopressin a fourfold potentiation of CRF activity with steep dose-response characteristics were observed. It also potentiated vasopressin-free hypothalamic extracts, suggesting that a new CRF does not account for all the nonvasopressin portion of the CRF complex.