We hypothesized that elevated macula densa intracellular (pHi) during tubuloglomerular feedback enhances O2- production from NAD(P)H oxidase. Microdissected thick ascending limbs from rabbit with intact macula densa were cannulated and perfused with physiological saline. When luminal NaCl was switched from 10 to 80 mM, O2- production increased from 0.53 ± 0.09 to 2.62 ± 0.54 units/min (p < 0.01). To determine whether inhibiting the Na/H exchanger blocks O2- production, we used dimethyl amiloride (DMA) to block Na/H exchange. In the presence of DMA, O2- production induced by NaCl was blunted by 40%. To study the effect of pHi on O2- in intact macula densa cells, we measured O2- while pHi was changed by adjusting luminal pH. When the macula densa was perfused with 80 mM NaCl and the pH of the perfusate was switched to 6.8, 7.4 and 8.0, O2- production was significantly enhanced, but not in 10 mM NaCl. To ascertain the source of O2-, we used the NAD(P)H oxidase inhibitor apocynin. In the presence of apocynin (10 -5 M), O2- production induced by elevating pHi was blocked. Finally, we measured the optimum pH for O2- production by the macula densa and found optimum extracellular pH is at 7.7 and optimum pHi is about 8 for O2- production. We found that elevated pHi enhances the O2- production from NAD(P)H oxidase induced by increasing luminal NaCl when the lumen is perfused with 80 mM NaCl, not 10 mM; and O2- production is pH-sensitive with an optimum pHi of 8.