Endothelin-1 inhibits sodium reabsorption in the thick ascending limb (THAL) via stimulation of nitric oxide (NO) production. The mechanism whereby endothelin-1 stimulates THAL NO is unknown. We hypothesized that endothelin-1 stimulates THAL NO production by activating phosphatidylinositol 3-kinase (PI3K), stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport. We measured 1) NO production by fluorescence microscopy using DAF2-DA, 2) Akt activity using a fluorescence resonance energy transfer-based Akt reporter, 3) phosphorylated NOS3 and Akt by Western blotting, and 4) NKCC2 activity by fluorescence microscopy. In isolated THAL, endothelin-1 (1 nmol/liter) increased NO production from 0.23 +/- 0.24 to 2.81 +/- 0.32 fluorescence units/min (p < 0.001; n = 5) but failed to stimulate NO production in THALs isolated from NOS3-/- mice. Wortmannin (150 nmol/liter), a PI3K inhibitor, reduced endothelin-1-stimulated NO by 83% (0.49 +/- 0.13 versus 3.31 +/- 0.49 fluorescence units/min for endothelin-1 alone; p < 0.006; n = 5). Endothelin-1 stimulated Akt activity by 0.16 +/- 0.02 arbitrary units as measured by fluorescence resonance energy transfer (p < 0.001; n = 5) and increased phosphorylation of Akt at Ser473 by 56 +/- 11% (p < 0.002; n = 7). Dominant-negative Akt blocked endothelin-1-induced NO by 60 +/- 8% (p < 0.001 versus control; n = 6), and an Akt inhibitor had a similar effect. Endothelin-1 increased phosphorylation of NOS3 at Ser1177 by 89 +/- 24% (p < 0.01; n = 7) but had no effect on Ser633. Endothelin-1 inhibited NKCC2 activity, an effect that was blocked by dominant-negative Akt and NOS inhibition. We conclude that endothelin-1 stimulates THAL NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport.