The aims of the current study were to determine whether superoxide (O₂^-) production is enhanced in mTAL of Dahl salt-sensitive (SS) rats compared to a salt-resistant consomic control strain (SS.13^BN) and to elucidate the cellular pathways responsible for augmented O₂^- production. Studies were carried out in 7-10 week old male SS and SS.13^BNrats fed either a 0.4% NaCl diet or a 4.0% NaCl diet for 3 days prior to tissue harvest. Tissue strips containing mTAL were isolated from the left kidney, loaded with the O₂^- sensitive fluorescent dye DHE, superfused with modified Hanks solution and imaged at X60 magnification on a heated microscope stage. O₂^- production was stimulated in mTAL by incrementing superfusate NaCl concentration from 154 to 254 to 500mM. O₂^- production was enhanced in mTAL of SS rats compared to SS.13^BNrats in response to incrementing bath NaCl. Addition of N-methyl-amiloride (100µM) or inhibition of NAD(P)H oxidase reduced O₂^- production in SS mTAL to levels observed in SS.13^BNrats. Both amiloride and ouabain sensitive pathways of O₂^- production were elevated following three days of high (4.0%) NaCl feeding in mTAL of SS and SS.13^BN rats. We conclude that mTAL from SS rats exhibit enhanced amiloride sensitive O₂^- production. The amiloride sensitive O₂^- response in mTAL is independent of active Na⁺ transport and appears to be mediated by NAD(P)H oxidase. Amiloride sensitive O₂^- production is likely to contribute to augmented outer medullary O₂^- production observed in SS rats during both normal and high NaCl diets.