Aminopeptidase A (APA) is a metalloprotease that is activated in cardiovascular diseases. Ang-(1-7) has been shown to have beneficial effects in myocardial injury. The aims of this study are to (1) Test the role of APA in cardiac pathology using the myocardial infarction (MI) model, (2) Investigate the role of APA in Ang-(1-7) metabolism (3) Explore the effect of APA inhibition on Ang-(1-7) homeostasis. APA activity was measured using mass spectrometry (MS) based in vitro and in situ enzyme assays. For the in vitro assay, heart homogenates were incubated with 50 μM Ang II in assay buffer and for absolute quantitation stable-isotope labeled internal standards were added. In the in situ MS imaging, frozen myocardial sections were incubated with 100 μM Ang II for 10 min and the resultant peptides were assessed on the tissue sections. For kinetic studies and Km determination, recombinant APA was incubated with Ang II and Ang-(1-7) in concentrations ranging from 1-100 μM. Results from the in vitro MS enzyme assays showed that myocardial APA activity was significantly increased after 24 hrs MI compared to sham (Ang III=0.82±0.02 vs. 0.32± 0.02 ρmol/min/μg in MI vs sham, respectively, p<0.01). The in situ MS imaging results confirmed that Ang III intensity was higher in MI heart sections compared to sham (96325±214 for MI and 1041±75 AU for Sham, p<0.05). Both MS enzyme assays identified a new peptide Ang-(2-7), m/z 784, which accumulated in the MI compared to Sham (136.96± 6.5 vs 78.94±3 AU in MI and Sham, respectively, p<0.05). Inhibition of APA with 4-Amino-phosphono-butyric acid significantly decreased formation of Ang-(2-7) from Ang-(1-7) and Ang III from Ang II by 98± 0.7% and 82±2.6% (p<0.05), respectively, indicating that APA is the responsible enzyme for the myocardial formation of both peptides. Interestingly, APA exhibited a higher substrate affinity for Ang-(1-7) compared to Ang II (Km for Ang II=14.32±1.6 vs Km for Ang-(1-7) =6.34± 1.12 μM, p<0.05). In conclusion, we have shown for the first time that APA is responsible for Ang-(1-7) degradation to Ang-(2-7). An upregulation of this pathway in myocardial injury may deprive the heart of cardioprotective Ang-(1-7). Thus, APA can be a potentially novel therapeutic target for management of myocardial remodeling after MI.