Superoxide(O₂^-) increases Na⁺ reabsorption in the thick ascending limb (THAL) by enhancing Na/K/2Cl cotransport. However, the effects of O₂^- on other THAL transporters, such as Na⁺/H⁺ exchangers, are unknown. We hypothesized that O₂^- stimulates Na⁺/H⁺ exchange in the THAL. We assessed total Na⁺/H⁺ exchange activity by measuring recovery of intracellular pH (pH_i) after acid loading in isolated perfused THALs before and after adding xanthine oxidase and hypoxanthine. We found that xanthine oxidase and hypoxanthine decreased total pH_i recovery rate from 0.26 ± 0.05 to 0.21 ± 0.04 pH units/min (p < 0.05), and this net inhibition decreased steady-state pH_i from 7.52 to 7.37. Because THALs have different Na⁺/H⁺ exchanger isoforms on the luminal and basolateral membrane, we tested the effects of xanthine oxidase and hypoxanthine on luminal and basolateral Na⁺/H⁺ exchange by adding dimethylamiloride (DMA) to either the bath or lumen. Xanthine oxidase and hypoxanthine increased luminal Na⁺/H⁺ exchange from 3.5 ± 0.8 to 6.7 ± 1.4 pmol/min/mm (p < 0.01), but decreased basolateral Na⁺/H⁺ exchange from 10.8 ± 1.8 to 6.8 ± 1.1 pmol/min/mm (p < 0.007). To ascertain whether these effects were caused by O₂^- or H₂O₂, we examined the ability of Tempol, a superoxide dismutase mimetic, to block these effects. In the presence of Tempol, xanthine oxidase and hypoxanthine had no effect on luminal or basolateral Na⁺/H⁺ exchange. We conclude that O₂^- inhibits basolateral and stimulates luminal Na⁺/H⁺ exchangers, perhaps due to the fact that different isoforms are expressed on each membrane. Inhibition of basolateral Na⁺/H⁺ exchange may enhance stimulation of luminal Na⁺/H⁺ exchange by providing additional protons to be extruded across the luminal membrane. Together, the effects of O₂^- on Na⁺/H⁺ exchange may increase net HCO₃^- reabsorption by the THAL.