The Na+/H+ Exchanger 3 (NHE3) plays an important role in the regulation of Na+and HCO3- reabsorption in proximal tubules of the kidney, but its contribution to the renal mechanisms of angiotensin II (ANG II)-induced hypertension remains unknown. Using global NHE3-deficient mice with transgenic rescue of the NHE3 gene in intestines (tgNhe3-/-) as a model of proximal tubule-specific Nhe3-/- mice, we tested the hypothesis that deletion of NHE3 selectively in proximal tubules impairs long-term blood pressure responses to extracellular and intracellular ANG II. Three groups of wild-type (tgNhe3+/+) and six groups of tgNhe3-/- mice (n=>8 per group) were treated with vehicle, ANG II (40 ng/min, i.p.), or in vivo adenovirus-mediated transfer of an intracellular ANG II fusion protein, ECFP/ANG II, for 2 weeks. Under basal conditions, tgNhe3-/- mice had significantly lower systolic blood pressure (SBP) (tgNhe3+/+: 119±3 mmHg vs. tgNhe3-/-: 109±3 mmHg, p<0.01), glomerular filtration rate (tgNhe3+/+: 148.7±13.0 vs. tgNhe3-/-: 83.9±5.1 μl/min, p<0.01), 24 h urine excretion (tgNhe3+/+: 1.25±0.15 vs. tgNhe3-/-: 0.68±0.10 ml, p<0.05), 24 h urinary Na+ excretion (tgNhe3+/+: 210.5.1±10.3 vs. tgNhe3-/-: 36.3±2.3 μmol, p<0.01), and 24 h urinary K+ excretion (tgNhe3+/+: 343.8±19.4 vs. tgNhe3-/-: 125.5±13.3 μmol, p<0.01). By contrast, basal plasma ANG II (tgNhe3+/+: 303.4±26.9 vs. tgNhe3-/-: 397.7±27.2 pg/ml, p<0.05), aldosterone (tgNhe3+/+: 506±26.3 vs. tgNhe3-/-: 759.9±12.7 pg/ml, p<0.01), and the expression of Na+/HCO3- cotransporter and Na+/K+-ATPase proteins in proximal tubules (p<0.01) were significantly increased in tgNhe3-/- mice. Deletion of NHE3 selectively in proximal tubules significantly attenuated the SBP responses to exogenous ANG II (tgNhe3+/+: 50±3 vs. tgNhe3-/-: 33±5 mmHg, p<0.01) and intracellular ECFP/ANG II (tgNhe3+/+: 18±3 vs. tgNhe3-/-: 6±2 mmHg, p<0.01) in tgNhe3-/- mice. 24 h urine and urinary Na+ excretory responses to ANG II were also attenuated in these mice (p<0.01), whereas ECFP/ANG II had no further effects on these responses. We concluded that NHE3 in proximal tubules of the kidney is required for maintaining long-term blood pressure responses to extracellular and intracellular ANG II and body salt and fluid homeostasis.