Superoxide (O(2)(-)) produced by NADPH oxidase regulates Na absorption and renal hemodynamics. Increased NaCl in the thick ascending limb (TAL) stimulates O(2)(-) generation. However, we do not know whether physiological changes in NaCl concentration augment O(2)(-) generation, nor do we know the mediator(s) involved. In other cells, Rac1, a regulatory subunit of NADPH oxidase, is activated by elevated NaCl. We hypothesized that increasing luminal NaCl within the physiological range activates Rac1 and NADPH oxidase and, thereby, increases O(2)(-) production. We increased NaCl from 10 to 57 mM in medullary TAL suspensions and used lucigenin to measure O(2)(-) generation and Western blot to measure Rac1 activity. Increasing NaCl stimulated O(2)(-) generation from 1.41 +/- 0.16 to 2.71 +/- 0.30 nmol O(2)(-) x min(-1) x mg protein(-1) (n = 6, P < 0.05). This increase was blocked by the Na-K-2Cl cotransporter inhibitor furosemide and the NADPH oxidase inhibitor apocynin. To examine the role of Rac1 in NaCl-induced O(2)(-) production, we measured Rac1 translocation by Western blot. When we added NaCl, Rac1 in the particulate fraction increased from 6.8 +/- 0.8 to 11.7 +/- 2.4% of total Rac1 (n = 7, P < 0.05). Then we measured O(2)(-) generation in the presence and absence of the Rac1 inhibitor. In the absence of the Rac1 inhibitor, NaCl increased O(2)(-) generation from 1.07 +/- 0.24 to 2.02 +/- 0.49 nmol O(2)(-) x min(-1) x mg protein(-1), and this increase was completely blocked by the inhibitor. Similarly, in vivo treatment of TALs with adenovirus expressing dominant-negative Rac1 decreased NaCl-induced O(2)(-) generation by 60% compared with control (0.33 +/- 0.04 vs. 0.81 +/- 0.17 nmol O(2)(-) x min(-1) x mg protein(-1), n = 6, P < 0.05). We concluded that physiological increases in NaCl stimulate TAL O(2)(-) generation by activating Rac1.