We previously observed that decreased brain ACE2 expression/activity contributes to the development of Angiotensin-II (Ang-II)-mediated hypertension in mice. To identify the mechanism involved in the impaired ACE2 expression/activity during hypertension, we first investigated ACE2 subcellular localization in the presence of Ang-II in vitro. Neuro2A cells (neuroblastoma) were transfected with a GFP-tagged human ACE2 plasmid construct (48 hours) and treated with Ang-II (100 nM, 4 hours). ACE2 localization was visualized with a flurorescence microscope (n=6), and protein levels on plasma membrane were determined by biotinylation followed by western blot (n=3). ACE2 expression was reduced at the plasma membrane and co-localized with the lysosomal marker Rab7 after Ang-II treatment, suggesting a translocation of ACE2 from plasma membrane to lysosomes. Biotinylation experiments confirmed a 50% reduction of plasma membrane ACE2 expression in Ang-II treated cells compared to vehicle group. To determine whether ACE2 internalization is responsible for the development of hypertension, C57Bl6 mice were implanted with telemetry probes for conscious blood pressure recording, and osmotic pumps for the following treatment groups: (n=6/group), 1) saline sc + aCSF icv; 2) Ang-II (600 ng/kg·min, sc, 14 days) + aCSF icv; 3) Ang-II + leupeptin (lysosomal inhibitor; 640 ng/kg·min, icv, 14 days); 4) saline sc + Leupeptin icv. After 2 weeks of treatment, leupeptin alone did not alter mean arterial blood pressure (MAP) compared to vehicle treatment (97±1 vs. 97±2 mmHg), however, it prevented Ang-II-mediated-increase in MAP (117±8 mmHg vs. 135±10 mmHg, P<0.01). To test whether the leupeptin-mediated prevention of hypertension involved ACE2, we infused ACE2 knockout mice with Ang-II sc in the presence or absence of leupeptin icv. Interestingly, Ang-II-mediated increase in MAP in ACE2 knockout mice (131±8 mmHg) was not significantly changed by icv leupeptin infusion (125±8 mmHg), suggesting that leupeptin prevented hypertension by inhibiting brain ACE2 degradation. Our data suggest that Ang-II promotes ACE2 internalization and degradation in lysosomes, while prevention of ACE2 degradation by leupeptin reverses the development of hypertension.