The effects of ovine CRF, lysine vasopressin (LVP), and their interrelationships, and rat hypothalamic extract (HME), on ACTH and beta-endorphin release by human pituitary tumor cells from two patients with Nelson's syndrome and one with Cushing's disease and on ACTH and cortisol secretion in vivo were studied. In cultured pituitary tumor cells, both LVP and CRF greatly stimulated ACTH and beta-endorphin release at maximally active concentrations of 0.1 microM and 10 nM, respectively. At these concentrations, the combination of the two substances had an additive or synergistic effect on hormone release. Low concentrations of HME potentiated and/or were synergistic with CRF-mediated ACTH release. In vivo, the combination of CRF (1 microgram/kg) and LVP (10 pressor units) induced greater ACTH release than the sum of the responses to CRF and LVP alone. This synergistic effect of CRF plus LVP concerned only ACTH release, while cortisol release after CRF plus LVP was equivalent to the sum of the maximal increments in this hormone after CRF and LVP alone. The peak levels of cortisol after a combination of CRF and LVP probably reflect the maximum stimulatory capacity of the adrenal cortex. These data support the concept that in man, both ovine CRF and vasopressin are corticotropin-releasing factors which act synergistically. Both substances might well regulate, at the pituitary level, the responsiveness of the pituitary-adrenal axis to stimuli reaching the hypothalamus. A test using ovine CRF and LVP together might provide a better index of total pituitary ACTH reserve than one using the two compounds separately.