Despite the plethora of data indicating beneficial effects of angiotensin (1-7) (Ang 1-7) on the cardiovascular system, its putative receptor, Mas, has not been characterized in tissue membrane preparations other than single concentration demonstrations of the localization of 125 I-Ang 1-7 binding sites in rat kidney. This does not indicate the specificity of 125 I-Ang 1-7 binding nor does it indicate the actual densities of the binding sites, i.e., B max (fmoles/mg tissue), or dissociation constant (K D ) to indicate binding affinity of 125 I-Ang 1-7 for its putative receptor. To characterize 125 I-Ang 1-7 binding in the kidney we prepared a low specific activity, monoradioiodinated Ang 1-7 using a 1:19 mix of 125 iodine : 127 iodine which allows for assessment of the B max and K D with concentrations of radioligand up to 100 nM. Frozen kidneys from adult male albino rats were dissected and homogenized in water and the membranes were precipitated by centrifugation at 48 kxG. Membranes were resuspended in Tris:MgCl 2 (50:1) pH 7.2 and incubated with 12 concentrations of 125/127 I-Ang 1-7 ranging from ~3-100 nM for 30 min at 22 C, after which bound 125/127 I-Ang 1-7 was resolved from unbound 125/127 I-Ang 1-7 by filtration and measured with a gamma counter. Specific binding (defined as 100 μM Ang 1-7 displaceable binding) of 125/127 I-Ang 1-7 showed a moderate binding affinity (K D = 14.7 ± 1.8 nM) and binding site density (B max = 24.5 ± 9.9 fmoles/mg initial wet weight). The B max value tended to be lower than that in the liver (B max = 62.3 ± 20.1 fmoles/mg initial wet weight) and the K D value was significantly greater (lower affinity) than that in the liver ( K D = 5.7 ± 0.6 nM, p = 0.0085). Of note, competition for 125/127 I-Ang 1-7 binding Ang 1-7 indicated that the IC 50 for Ang 1-7 competition for 125/127 I-Ang 1-7 binding was 42.5 μM. Moreover, the ability of a variety of angiotensin peptides to inhibit 125/127 I-Ang 1-7 binding at 100 μM, Ang 1-7 was less potent that the other angiotensin peptides: Ang III > Ang II > Ang I ~ Ang IV > Ang 2-7 > Ang 1-7 ~ Ang 3-7. These studies suggest that the binding site for 125/127 I-Ang 1-7 is not specific for the putative Ang 1-7 receptor mas, and may represent a low affinity binding to the AT 1 or AT 2 receptor