Multiple isoforms of NADPH oxidases (Nox) have been identified in the kidney including Nox1, Nox2, and Nox4 which contribute to elevated levels of renal oxidative stress in hypertension. However, the relative contribution and the mechanisms of their regulation of these specific isoforms remain unclear. The role of Nox4 has received the least attention even though it is the most highly expressed isoform in the kidney. It is also unique in that it is thought to release primarily H2O2. In previous studies using Sprague Dawley rats, we found that increased luminal flow and Na+ delivery increased intracellular H2O2 production in epithelial cells of the medullary thick ascending limb of Henle (mTAL). In the present study, we determined whether Nox4 contributed to the flow induced increases of mTAL H2O2. As part of a broader study, microdissected-isolated mTAL from Dahl salt-sensitive (SS) rats fed a 0.4% NaCl since weaning were examined and the presence of Nox4 mRNA confirmed. Ex vivo studies were then performed to determine if absence of Nox4 reduced H2O2 production in response to increases of mTAL luminal perfusion from 5 to 20 nl/min. Isolated microperfused mTAL tubular responses of SS rats fed a 4% NaCl diet for 3 days were compared to SS Nox4 null mutant rats (SSNox4-/-) which were created using zinc finger nuclease techniques. A sensitive intracellular fluorescent probe (PF6-AM), highly specific to H2O2,was used to quantify responses to luminal flow. Results showed that H2O2 production following the increase in luminal perfusion rate was reduced in SSNox4-/- rats nearly 85% compared to SS (1300±138 Fluorescent Units (FU); n=5) versus 244±185 FU (n=8; p<0.05). We conclude that Nox4 is of major importance in the production of H2O2 in response to an increase in luminal flow and Na+ delivery in the mTAL of SS rats and could represent an important therapeutic target for salt-sensitive hypertension.