Abstract: 125I-Bolton-Hunter (125I-BH) neuropeptide Y (NPY) was used to identify specific high-affinity NPY binding sites in porcine aortic smooth muscle membrane fractions and to characterize the binding sites in comparison with those in porcine hippocampal membrane fractions. Ca2+, but not Mg2+ or Mn2+, enhanced specific 125I-BH-NPY binding in aortic smooth muscle, while all of the cations did in hippocampus. The fast, saturable, and selective binding was to a single population of sites, with a KD of 0.99 ± 0.11 nM and a Bmax of 0.35 ± 0.06 fmol/mg protein. GTP and its non-hydrolyzable analogues reduced 125I-BH-NPY binding dose dependently. Neither calcium channel blockers nor nor-adrenaline had any effect on the binding. Among structurally related peptides, peptide YY displaced 125I-BH-NPY binding as potently as NPY, while human, avian, and rat pancreatic polypeptides displaced 125I-BH-NPY binding 100 times less potently than NPY. The C-terminal fragment NPY(13–36) for inhibiting 125I-BH-NPY binding in aortic smooth muscle was ∼40 times less potent than in the hippocampus. When we examined the effect of these peptides on cytosolic free Ca2+ ([Ca2+]i) in cultured porcine aortic smooth muscle cells, only the peptides showing high affinity for 125I-BH-NPY binding sites increased [Ca2+]i. These results indicate that the NPY binding sites labeled by 125I-BH-NPY in aortic smooth muscle are functional receptors and have different properties from those in the hippocampus with respect to dependency on divalent cations, sensitivity to GTP analogues, and affinity for NPY(13–36) and pancreatic polypeptides.