We showed that luminal flow increases net superoxide (O(2)(-)) production via NADPH oxidase in thick ascending limbs. Protein kinase C (PKC) activates NADPH oxidase activity in phagocytes, cardiomyocytes, aortic endothelial cells, vascular smooth muscle cells, and renal mesangial cells. However, the flow-activated pathway that induces NADPH oxidase activity in thick ascending limbs is unclear. We hypothesized that PKC mediates flow-stimulated net O(2)(-) production by thick ascending limbs. Initiation of flow (20 nl/min) increased net O(2)(-) production from 4 +/- 1 to 61 +/- 12 AU/s (P < 0.007; n = 5). The NADPH oxidase inhibitor apocynin completely blocked the flow-induced increase in net O(2)(-) production (2 +/- 1 vs. 1 +/- 1 AU/s; P > 0.05; n = 5). Flow-stimulated O(2)(-) was also blocked in p47(phox)-deficient mice. We measured flow-stimulated PKC activity with a fluorescence resonance energy transfer (FRET)-based membrane-targeted PKC activity reporter and found that the FRET ratio increased from 0.87 +/- 0.02 to 0.96 +/- 0.04 AU (P < 0.05; n = 6). In the absence of flow, the PKC activator phorbol 12-myristate 13-acetate (200 nM) enhanced net O(2)(-) production from 5 +/- 2 to 92 +/- 6 AU/s (P < 0.001; n = 6). The PKC-alpha- and betaI-selective inhibitor Gö 6976 (100 nM) decreased flow-stimulated net O(2)(-) production from 54 +/- 15 to 2 +/- 1 AU/s (P < 0.04; n = 5). Flow-induced net O(2)(-) production was inhibited in thick ascending limbs transduced with dominant-negative (dn)PKC-alpha but not dnPKCbetaI or LacZ (Delta = 11 +/- 3 AU/s for dnPKCalpha, 55 +/- 7 AU/s for dnPKCbetaI, and 63 +/- 7 AU/s for LacZ; P < 0.001; n = 6). We concluded that flow stimulates net O(2)(-) production in thick ascending limbs via PKC-alpha-mediated activation of NADPH oxidase.