Abstract—Superoxide (O₂^-) is an important regulator of kidney function. We have recently shown that luminal flow stimulates O₂^- production in the thick ascending limb (TAL), attributable in part to mechanical factors. Stretch, pressure and shear stress all change when flow increases in the TAL. We hypothesized that stretch rather than shear stress or pressure per se stimulates O₂^- production by TALs. We measured O₂^- production in isolated perfused rat TALs using fluorescence microscopy and dihydroethidium. Tubules were perfused with a Na-free solution to eliminate the confounding effect of Na transport. Flow induced an increase in O₂^- production from 29±4 to 90±8 AU/s (P<0.002; n=5). The response to flow is rapidly reversible. O₂^- production by TALs perfused at 10 nL/min decreased from 113±6 to 25±10 AU/s (P<0.003; n=4) 15 minutes after flow was stopped. Increasing pressure and stretch in the absence of shear stress caused a significant increase in O₂^- production (40±6 to 118±17 AU/s; P<0.02; n=5). In contrast, eliminating shear stress had no effect (107±9 versus 108±10 AU/s; n=5). Increasing stretch by 27±2% in the presence of flow while reducing pressure stimulated O₂^- production from 66±7 to 84±9 AU/s (29±8%; P<0.02; n=5). Tempol inhibited this increase (n=5). We conclude that increasing stretch rather than pressure or shear stress accounts for the mechanical aspect of flow-induced O₂^- production in the TAL. Stretch of the TAL during hypertension, diabetes, and salt loading may contribute to renal damage.