To investigate the mechanism by which ACTH secretion is inhibited during hypothermia, hypophysial portal blood was collected from euthermic and hypothermic rats, and the concentrations of corticotropin-releasing factor (CRF), vasopressin (AVP) and oxytocin (OT) were measured by radioimmunoassay. CRF levels in portal plasma were not different in the 2 groups; AVP and OT levels were significantly lower in hypothermic rats. The concentration of AVP and OT in peripheral plasma was also significantly lower in hypothermic rats compared with euthermic controls. The pituitary responsiveness to CRF during hypothermia was tested in vivo and in vitro. In pentobarbital-anesthetized male rats injected i.v. with 0.1 or 1.0 nmol CRF, the ACTH response was significantly smaller in hypothermic compared with euthermic animals. Hemipituitaries superfused at 31.degree. C released the same amount of ACTH in response to 1 nM CRF as hemipituitaries superfused at 37.degree. C (31.degree. C, 541 .+-. 90 pg; 37.degree. C, 563 .+-. 29 pg) despite reduced baseline secretion (31.degree. C, 77 .+-. 10 pg/10 min; 37.degree. C, 114 .+-. 14 pg/10 min; P < 0.05). Apparently, the inhibition of ACTH secretion during hypothermia is mediated by decreased hypothalamic secretion of AVP and OT which in turn decreases the pituitary responsiveness to CRF.