Nox4 and Nox2 are the most abundant NADPH oxidases (Nox) in the kidney and have been shown to contribute to hypertension, renal oxidative stress and injury in Dahl salt-sensitive (SS) hypertensive rats. The present study focused on the role of Nox4 and p67phox/Nox2 on the generation of H2O2 and O2•- in mTAL of SS rats in response to increasing luminal flow (5 to 20 nl/min). Nox4 and p67phox/Nox2 genes were found to be expressed in the renal medullary thick ascending limb of Henle (mTAL) of SS rats. Responses of SS rats were compared to SS rats with knockout of Nox4 (SSNox4-/-) or functional mutation of p67phox (SSp67phox-/-). Results show that Nox4 was the dominant source of increased intracellular H2O2 production in response to increased luminal flow as determined with the fluorescent dye PF6-AM. The rate of mitochondrial H2O2 production (mito-PY1) was also significantly reduced in SSNox4-/- compared to SS rats but not in SSp67phox-/- rats. In contrast, intracellular O2•- production (Eth/DHE) in mTAL of SSNox4-/- rats was nearly identical to SS rats in response to luminal flow indicating Nox4 made no measurable contribution. SSp67phox-/- rats exhibited reduced mTAL O2•- production compared to SS rats at the lower luminal flow of 5 nl/min and progressively increased when perfusion was changed to 20 nl/min. We conclude that increased mTAL luminal flow results in increases of both intracellular and mitochondrial H2O2 which are dependent upon the presence of Nox4 and that p67phox/Nox2 accounts solely for increases of O2•- production.