Abstract—The effects of NaCl, glucose, and thyroid hormone on the production of superoxide (O2·−) within the renal medulla of Sprague-Dawley rats were examined. Responses of intracellular superoxide [O2·−]iin isolated medullary thick ascending limbs (mTALs) were studied using real-time fluorescent microscopy with measurement of the dehydroethidium (DHE) to ethidium (Eth) conversion ratio (Eth/DHE ratio unit). The results demonstrated that elevations of extracellular NaCl (from 152 to 252 mmol/L), d-glucose (from 5 to 25 mmol/L), and triiodo-thyronine (T3; 10 &mgr;mol/L) significantly increased [O2·−]ilevels. Preincubation with superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid (1 mmol/L) significantly inhibited these responses. Stimulation with equamolar amounts of choline chloride or l-glucose failed to increase [O2·−]i, indicating that these O2·−responses were not determined by changes in osmolality. The responses to NaCl, d-glucose, and T3 were abolished by pretreatment with the Na+/K+-ATPase pump inhibitor ouabain (4 mmol/L) and with Na+/H+-exchanger inhibitor dimethylamiloride (100 &mgr;mol/L). We conclude that elevations of extracellular NaCl, d-glucose, or T3 levels can activate both the Na+/K+-ATPase pump and Na+/H+exchanger in mTAL, which, in turn, is associated with increased intracellular concentrations of superoxide.