A molecular imaging technique was established to monitor tissue specific processing of the vasoconstrictor peptide, angiotensin II (Ang II). Mouse kidney sections were incubated with Ang II followed by analysis with MALDI (matrix assisted laser desorption/ionization) imaging. The method allows for the spatial and molecular characterization of peptide processing. Using this technique, conversion of Ang II to Ang III mainly occurred in the renal medulla. Ang (1–7) was predominantly formed in renal cortex as was Ang (1–4), a tetrapeptide generated from Ang (1–7). These results were verified with enzyme assays using tissue punches of renal medulla and cortex. Ang III and Ang (1–4) formation were comparable in Ang converting enzyme 2 (ACE2) knockout (KO) and wild type (WT) mice. Ang (1–7) formation was lower in ACE2KO, but still present. Using recombinant enzymes, we determined that two other peptidases, prolyl carboxypeptidase (PCP) and prolyl endopeptidase (PEP), are capable of converting Ang II to Ang (1–7). Incubation of kidney sections with a PCP/PEP inhibitor reduced Ang (1–7) formation in WT and ACE2KO suggesting a contribution of PCP/PEP to ACE2 independent Ang (1–7) generation in kidney. This novel molecular imaging technique can be expanded to assess actions and catalytic specificity of other enzymes in mammalian tissues. Supported by NIH R01HL093567 and F32DK093226-01.