Arginine-vasopressin (AVP) is regarded as a potent stimulator of pituitary adrenocorticotropin (ACTH) secretion and participates therefore in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis function in concert with the physiological activator of the axis, hypothalamic corticotropin-releasing hormone (CRH). We examined the effects of AVP and/or three synthetic V_1b receptor antagonists on the activity of the HPA axis in vivo and in vitro in the rat. AVP was injected intravenously to Sprague-Dawley rats (1 µg/rat) through an indwelling jugular catheter. AVP stimulated ACTH release, with maximal effect 10 min after injection. Intravenous injection of three V_1b antagonists, [1-(β-mercapto-β, β-cyclopentamethylenepropionic acid),2-O-ethyltryrosine,4-valine] arginine vasopressin (d(CH2)s[Tyr(Et²)]VAVP (WK 1-1), 9-desglycine[1-(β-mercapto-β, β-cyclopentamethylenepropionic acid),2-O-ethyltyrosine,4-valine] arginine vasopressin desGly⁹d(CH2)5[Tyr(Et²)]-VAVP (WK 3-6), and 9-desglycine[1-(β-mercapto-β, β-cyclopentamethylene-propionic acid),2-D-(0-ethyl)tyrosine,4-valine] arginine vasopressin des Gly₉d(CH₂)₅[D-Tyr(Et²)]VAVP (AO 3-21), prevented AVP-stimulated ACTH secretion. Explanted rat hypothalami incubated in vitro with graded concentrations of AVP (10-¹⁴-10-⁵M) secreted immunoreactive CRH (iCRH) in a concentration-dependent fashion. Maximal stimulatory effect occurred at the concentration of 10-⁶M. Incubation of hypothalami with WK 1-1, WK 3-6, or AO 3-21 (10-⁶M) prevented AVP-stimulated iCRH secretion. Results suggest that AVP plays a relevant, multiple role in the activation of the HPA axis in the rat. In fact, interactions between AVP and the pituitary corticotroph may not be solely due to a direct effect of AVP on ACTH secretion, but also to activation of the hypothalamic CRH neuron. These effects of AVP appear to be mediated by the V_1b receptor subtype.