In the domestic fowl, angiotensin (ANG) II causes a unique vasodepressor response in vivo and vascular relaxation of aortic rings in vitro that appear to be mediated by ANG II receptors. In initial studies using radioligand binding techniques, we identified specific vascular ANG II receptors in the fowl aorta. In the present study, we have characterized fowl vascular ANG II receptors in terms of binding specificity and their modulation by divalent cations and guanine nucleotide, to understand how the fowl receptor might differ from mammalian vascular ANG II receptors that mediate vasoconstriction. Competitive displacement of [125I] ANG II binding by ANG agonist and antagonist analogs revealed a unique pattern of receptor specificity, with the potency rank order: [Asn1, Val5]ANG II greater than [Asp1, Ile5]ANG II greater than [Asp1, Val5, Ser9] ANG I = [Asp1, Val5]ANG II much greater than [Val5]ANG III greater than [sarcosine(Sar)1, Ile5]ANG II greater than [Sar1, Ile8]ANG II much greater than [Sar1, Thr8]ANG II. Divalent cations (Ca++, Mg++ and Mn++) inhibited equilibrium radioligand binding by as much as 50% at 100 mM, with the potency order: Ca++ greater than Mn++ greater than Mg++. Mg++ and Mn++ stimulated binding very slightly (110%) at low doses (1-10 mM). The stable guanine nucleotide analog 5'-guanylyl imidodiphosphate inhibited equilibrium radioligand binding moderately (15% at 100 microM) in the presence of 10 mM MgCl2, but failed to alter the dissociation rate of receptor-bound ligand (half-time = 10.92 min). These results suggest that fowl vascular ANG II receptors exhibit specificity and regulatory properties fundamentally different from those of mammalian vascular ANG II receptors.