Hypertension is associated with increased levels of oxidative stress and medullary renal injury. Previous studies have shown that elevations in renal perfusion pressure increase Na(+) delivery to the medullary thick ascending limb (mTAL), and enhancement of NaCl transport in the outer medulla has been reported in many experimental forms of hypertension. This study examined the effects of increased Na(+) and fluid delivery in mTAL perfused in vitro on the generation of superoxide. Osmolality was maintained constant between low- and high-Na(+) perfusates by adjusting with choline Cl(-). Real-time fluorescent microscopic techniques were used to determine the generation of superoxide and nitric oxide in individual mTAL cells using dihydroethidium and DAF-FM dyes, respectively. Increasing the Na(+) concentration of the perfusate from 60 to 149 mM or luminal flow rate from 5 to 20 nl/min (with fixed Na(+) concentration of 60 mM) significantly increased superoxide generation and decreased nitric oxide in mTAL. These effects were inhibited when active transport of Na(+) was inhibited by ouabain. We conclude that increases in luminal Na(+) concentration and/or flow rate can increase the generation of superoxide in mTAL and reduce nitric oxide bioavailability. This may lead to reduction in medullary blood flow and promote hypoxia and tubular necrosis within the renal medulla during in hypertension.