In the microvillar microdomain of the kidney brush border, sodium hydrogen exchanger type 3 (NHE3) exists in physical complexes with the serine protease dipeptidyl peptidase IV (DPPIV). The purpose of this study was to explore the functional relationship between NHE3 and DPPIV in the intact proximal tubule in vivo. To this end, male Wistar rats were treated with an injection of the reversible DPPIV inhibitor Lys [Z(NO₂)]-pyrrolidide (I40; 60 mg·kg⁻¹·day⁻¹ip) for 7 days. Rats injected with equal amounts of the noninhibitory compound Lys[Z(NO₂)]-OH served as controls. Na⁺-H⁺exchange activity in isolated microvillar membrane vesicles was 45 ± 5% decreased in rats treated with I40. Membrane fractionation studies using isopycnic centrifugation revealed that I40 provoked redistribution of NHE3 along with a small fraction of DPPIV from the apical enriched microvillar membranes to the intermicrovillar microdomain of the brush border. I40 significantly increased urine output (67 ± 9%; P < 0.01), fractional sodium excretion (63 ± 7%; P < 0.01), as well as lithium clearance (81 ± 9%; P < 0.01), an index of end-proximal tubule delivery. Although not significant, a tendency toward decreased blood pressure and plasma pH/HCO₃⁻was noted in I40-treated rats. These findings indicate that inhibition of DPPIV catalytic activity is associated with inhibition of NHE3-mediated NaHCO₃reabsorption in rat renal proximal tubule. Inhibition of apical Na⁺-H⁺exchange is due to reduced abundance of NHE3 protein in the microvillar microdomain of the kidney brush border. Moreover, this study demonstrates a physiologically significant interaction between NHE3 and DPPIV in the intact proximal tubule in vivo.