The role of oxytocin (OT) in regulating stress-induced ACTH secretion was investigated by immunoneutralization of endogenous OT with an antiserum raised against synthetic OT. Rats were subjected to one of three stresses: novel environment, tail-hang, or ether. In otherwise untreated rats, ACTH levels rose at least 3-fold in response to all three stresses whereas OT levels increased only in response to tail-hang and ether. Injection of a highly specific antiserum to OT 60 min before the experiment inhibited the ACTH response to tail-hang and ether by 64 and 56%, respectively, but had no effect on the ACTH response to novel environment stress. The data support a physiologic role for OT in the control of ACTH secretion but suggest that it is not until OT levels rise in response to stress that the effects of OT are expressed.