The brain is thought to exert a predominantly stimulatory action on ACTH secretion mediated mainly by corticotrophin-releasing factor-41 (CRF-41) and arginine vasopressin (AVP). Several data, however, also point to the existence of an ACTH-inhibiting factor. Atrial natriuretic peptide (ANP), at concentrations found in hypophysial portal blood, inhibits ACTH release in vitro. The aim of the present studies was to use ANP immunoneutralization to determine whether ANP does in fact inhibit ACTH release in vivo. Intracerebroventricular infusion (I μl/min for 30 min) of sheep anti-ANP serum into male rats anaesthetized with sodium pentobarbitone had no significant effect on jugular venous plasma concentrations of ACTH or LH but did decrease significantly the plasma concentrations of prolactin. Intravenous infusion of 0.8 ml sheep anti-ANP serum but not control (non-immune) sheep serum, through an indwelling intra-atrial cannula in conscious male rats resulted in a marked and significant increase in plasma ACTH and corticosterone concentrations. The ACTH and corticosterone response to a 30-s ether stress was not significantly potentiated in the same conscious rats infused with anti-ANP serum. Intra-atrial infusion of anti-ANP did not significantly affect plasma prolactin, LH, glucose or sodium concentrations or plasma osmolality. These results show for the first time that ANP is a potent inhibitor of ACTH secretion in the conscious male rat and that, therefore, ANP is a hypothalamic neurohormone which is likely to play an important inhibitory role in the neural control of ACTH release.