Superoxide (O₂^-) regulates renal function and is implicated in hypertension. O₂^- production increases in response to increased ion delivery in thick ascending limbs (TALs) and macula densa, and mechanical strain in other cell types. Tubular flow in the kidney acutely varies causing changes in ion delivery and mechanical stress. We hypothesized that increasing luminal flow stimulates O₂^- production by NADPH oxidase in TALs via activation of Na/K/2Cl cotransport. We measured intracellular O₂^- in isolated rat TALs using dihydroethidium in the presence and absence of luminal flow and inhibitors of NADPH oxidase, Na/K/2Cl cotransport and Na/H exchange. In the absence of flow, the rate of O₂^- production was 5.8±1.4 a.u./sec. After flow was initiated, it increased to 29.7±4.3 a.u./sec (p<0.001). O₂^- production was linearly related to flow. Tempol alone and apocynin alone blocked the flow-induced increase in O₂^- production (3.5±1.7 vs. 4.5±2.8 a.u./sec and 8.2±2.1 vs. 10.6±2.8 a.u./sec, respectively). Furosemide decreased flow-induced O₂^- production by 55% (37.3±5.2 to 16.8±2.8 a.u./sec; p<0.002); however, dimethylamiloride had no effect. Finally, we examined whether changes in mechanical forces are involved in flow-induced O₂^- production by using a Na-free solution to perfuse TALs. In the absence of NaCl, luminal flow enhanced O₂^- production (1.5±0.5 to 13.5±1.1 a.u./sec; p<0.001), approximately 50% less stimulation than when flow was increased in the presence of luminal NaCl. We conclude that flow stimulates O₂^- production in TALs via activation of NADPH oxidase, and that NaCl absorption due to Na/K/2Cl cotransport and flow-associated mechanical factors contribute equally to this process.