To better understand the tissue distribution and activity of enzymes involved in angiotensin II (Ang II) processing, we developed a novel molecular imaging method using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Mouse kidney sections (12 μm) were incubated with 10–1,000 μmol/l Ang II for 5–15 min at 37°C. The formed peptides Ang III and Ang-(1–7) were identified by MALDI-TOF/TOF. A third metabolite, Ang-(1–4), was generated from further degradation of Ang-(1–7). Enzymatic processing of Ang II was dose and time dependent and absent in heat-treated kidney sections. Distinct spatial distribution patterns (pseudocolor images) were observed for the peptides. Ang III was localized in renal medulla, whereas Ang-(1–7)/Ang-(1–4) was present in cortex. Regional specific peptide formation was confirmed using microdissected cortical and medullary biopsies. In vitro studies with recombinant enzymes confirmed activity of peptidases known to generate Ang III or Ang-(1–7) from Ang II: aminopeptidase A (APA), Ang-converting enzyme 2 (ACE2), prolyl carboxypeptidase (PCP), and prolyl endopeptidase (PEP). Renal medullary Ang III generation was blocked by APA inhibitor glutamate phosphonate. The ACE2 inhibitor MLN-4760 and PCP/PEP inhibitor Z-pro-prolinal reduced cortical Ang-(1–7) formation. Our results establish the power of MALDI imaging as a highly specific and information-rich analytical technique that will further aid our understanding of the role and site of Ang II processing in cardiovascular and renal pathologies.