The present study was designed to test the hypothesis that the production of superoxide (O(2)-* by NAD(P)H oxidase is coupled to tubular metabolic activity through ionic activation mediated by H(+) movement across cell membrane. Using dual fluorescent microscopic imaging analysis, intracellular O(2)-* levels and pH (pH(i)) in renal medullary thick ascending limb of Henle (TALH) cells were simultaneously measured. It was found that intracellular O(2)-* levels in these cells were increased in parallel to the elevation of pH(i) by outflow of H(+) induced via NH(4)Cl loading followed by rapid removal. This increase in intracellular O(2)-* levels was substantially blocked by an inhibitor of Na(+)/H(+) exchanger, methylisobutyl-amiloride (MIA; 100 microM), a chemical SOD mimetic, Tiron (1 mM) or an inhibitor of NAD(P)H oxidase, diphenylene iodonium (DPI; 100 microM). In additional groups of TALHs, a proton ionophore, carbonylcyanide m-chlorophenylhydrazone (10 microM) was used to produce H(+) conductance, leading to H(+) flux across cell membrane depending on extracellular pH. The efflux of H(+) increased both pH(i) and intracellular O(2)-* levels, but the influx of H(+) did not increase intracellular O(2)-* levels. The H(+) efflux-induced increase in intracellular O(2)-* levels was completely blocked by DPI and another NAD(P)H oxidase inhibitor, apocynin (100 microM). In in invo experiments, renal medullary infusion of MIA (100 microM) was found to significantly decrease the concentrations of H(2)O(2) in the renal medullary interstitium. These results suggest that it is the outward movements of H(+) ions that activates NAD(P)H oxidase to produce O(2)-* in TALH cells. This H(+) outflow-associated activation of NAD(P)H oxidase importantly contributes to tissue levels of reactive oxygen species in the renal medulla.