The ACE‐2/angiotensin 1–7/Mas axis antagonizes many pathophysiological effects of the classical renin angiotensin system. There is considerable interest in measuring ACE‐2 and determining its tissue localization. Toward this end we have developed a small molecule ACE‐2 inhibitor that can be radioiodinated to assess the localization of ACE‐2 by in vitro autoradiography. This ACE‐2 inhibitor reduced metabolism of the synthetic ACE‐2 substrate Mca‐APK(Dnp) in the micromolar range. The ACE‐2 inhibitor was radioiodinated using the chloramine T procedure and the mono 125 I‐ACE‐2 inhibitor was purified by reverse phase HPLC. The radioligand displayed saturable binding to rat kidney homogenate that was displaceable by EDTA (10 mM). The radioligand (50 pM) was incubated with 20 micron –thick coronal sections of mouse brain in the presence of captopril (1 μM) with or without the peptidic human ACE‐2 inhibitor DX‐600 (1.3 μM). An alternate set of sections was incubated with captopril (1 μM) and EDTA. There was a diffuse pattern of 125 I‐labeled ACE‐2 inhibitor binding throughout the mouse brain. Binding in the presence to DX‐600 was only moderately decreased, however, it was almost completely eliminated in the presence of EDTA. These results indicate the utility of a radioiodinated ACE‐2 inhibitor for characterization and localization of ACE‐2. Supported in part by JFS Sales and Consulting, LLC.