To study the physiology of the high affinity vascular smooth muscle receptor for angio-tensin II, we have characterized125I-angiotensin II binding sites in a participate fraction prepared from rat mesenteric arteries.1251-Angiotensin II binding was saturable at physiological concentrations of hormone, and was of high affinity. Scatchard analysis indicated a single class of binding sites with an equilibrium dissociation constant (Kd) of 0.91 ± 0.11 (SD) nM, and a total binding capacity of 53.7 ± 3.0 fmol/mg protein. Parallel studies with3H-angiotensin II yielded a similar Kj (1.18 ± 0.48 nM) and total binding capacity (56.8 ± 9.2 fmol/mg protein).mI-Angiotensin II associated with binding sites rapidly (t1/2for association = 4 minutes at 37°C), and reversibly. Kinetic analysis of binding at 22°C by two independent methods yielded comparable values for both the association rate (4.0 and 6.8 x 105/M per sec) and dissociation rate (3.2 and 3.8 x 10−4sec) constants. Equilibrium dissociation constants calculated from kinetic analysis (0.56 and 0.80 nM) were in close agreement with that obtained from steady state Scatchard analysis. Analogues and antagonists of angiotensin II competed for binding in a potency series which exactly paralleled that observed for bioassay systems utilizing pressor response in vivo and vascular smooth muscle contraction in vitro.125I-Angiotensin II binding was stimulated 2-to 3-fold in the presence of 1 nun divalent cations (Mn2+> Mg2+> Ca2+) and reversibly inhibited by EDTA and EGTA. Dithiothreitol (5 mil), a sulfhydryl-reducing agent that has been reported to block vasoconstrictor response to angiotensin II, inhibited125I-angiotensin II binding by 45%. The present study defines specific angiotensin II binding sites in a muscular artery representative of the resistance vasculature. We conclude that these binding sites, unlike those previously described in conductance-type vessels (aorta), have a sufficiently high affinity to interact with physiological levels of hormone.Circ Res 47; 278-286 1980