The octapeptide, angiotensin II, elicits a positive inotropic response in myocardial tissue by activating slow calcium channels. Pharmacologic studies suggest that the inotropic action of angiotensin II is receptor mediated. The current investigation was performed to characterize the binding of 125I-angiotensin II to the putative receptor in a plasma membrane-sarcoplasmic reticulum preparation of the rabbit left ventricle. In experiments performed at 18°C, steady state binding occurred at 45 minutes and saturable binding was 80-85% of the total binding. Analysis of the binding data indicated that the 125I-angiotensin II interacted with a single class of sites with a K(d) = 4.5 ± 0.8 nM and exhibited a binding capacity of 53.5 ± 8 fmol/mg protein. The potency order for the competitive binding of analogues and antagonists of angiotensin II paralleled that observed for in vitro contractile force development in bioassay systems utilizing left atrial tissue. The binding of 125I-angiotensin II was stimulated 2-fold in the presence of the divalent cations of calcium and magnesium (10 mM). Guanine nucleotides modified the binding of 125I-angiotensin II to a rabbit myocardial particulate fraction. Guanine triphosphate and nonhydrolyzable analogues of guanine triphosphate increased the dissociation rate of the bound 125I-angiotensin II and decreased hormone binding to the receptor at equilibrium. In the absence of magnesium, guanine nucleotides had no effect on the dissociation rate of 125I-angiotensin II. 125I-Angiotensin II binding to the rabbit myocardial particulate fraction was found to have high affinity, to be saturable, reversible, specific, and modulated by guanine nucleotides. The characteristics of the binding site of 125I-angiotensin II indicate that it is the hormone receptor which mediates the physiological (inotropic) effects of angiotensin II in rabbit myocardial tissue.