Abstract—It has been reported previously that H⁺ efflux via the Na⁺/H⁺ exchange stimulates NAD(P)H oxidase–dependent superoxide (O₂^·-) production in medullary thick ascending limb. We have demonstrated recently that N-methyl-amiloride–sensitive O₂^·- production is enhanced in the thick ascending limb of Dahl salt-sensitive (SS) rats, suggesting that H⁺ efflux through Na⁺/H⁺ exchangers may promote renal oxidative stress and the development of hypertension in these animals. In the current study we demonstrate, using selective and potent inhibitors, that inhibition of Na⁺/H⁺ exchange does not mediate the ability of N-methyl-amiloride to inhibit thick ascending limb O₂^·- production. To determine the mechanism of action of N-methyl-amiloride, we examined H⁺ efflux and O₂^·- production in SS and SS.13^BN thick ascending limbs of prehypertensive, 0.4% NaCl–fed rats. Tissue strips containing the medullary thick ascending limb were isolated from male SS and salt-resistant consomic SS.13^BN rats, loaded with either dihydroethedium or 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, and imaged in a heated tissue bath. In Na⁺-replete media, activation of Na⁺/H⁺ exchange using an NH₄Cl prepulse did not stimulate thick ascending limb O₂^·- production. In Na⁺-free media containing BaCl₂ in which Na⁺/H⁺ activity was inhibited, an NH₄Cl prepulse stimulated O₂^·- production in medullary thick ascending limb renal tubular segments. This response was enhanced in medullary thick ascending limb of SS rats (slope ethidium/dihydroethedium=0.029±0.004) compared with SS.13^BN rats (slope=0.010±0.004; P<0.04) and could be inhibited by N-methyl-amiloride (slope=0.005±0.002 and 0.006±0.002 for SS and SS.13^BN, respectively). We concluded that only H⁺ efflux through a specific, as-yet-unidentified, amiloride-sensitive H⁺ channel promotes O₂^·- production in the medullary thick ascending limb and that this channel is upregulated in SS rats.