Angiotensin II (ANG II) stimulates production of superoxide (O₂⁻) by NADPH oxidase (NOX) in medullary thick ascending limbs (TALs). There are three isoforms of the catalytic subunit (NOX1, 2, and 4) known to be expressed in the kidney. We hypothesized that NOX2 mediates ANG II-induced O₂⁻ production by TALs. To test this, we measured NOX1, 2, and 4 mRNA and protein by RT-PCR and Western blot in TAL suspensions from rats and found three catalytic subunits expressed in the TAL. We measured O₂⁻ production using a lucigenin-based assay. To assess the contribution of NOX2, we measured ANG II-induced O₂⁻ production in wild-type and NOX2 knockout mice (KO). ANG II increased O₂⁻ production by 346 relative light units (RLU)/mg protein in the wild-type mice (n = 9; P < 0.0007 vs. control). In the knockout mice, ANG II increased O₂⁻ production by 290 RLU/mg protein (n = 9; P < 0.007 vs. control). This suggests that NOX2 does not contribute to ANG II-induced O₂⁻ production (P < 0.6 WT vs. KO). To test whether NOX4 mediates the effect of ANG II, we selectively decreased NOX4 expression in rats using an adenovirus that expresses NOX4 short hairpin (sh)RNA. Six to seven days after in vivo transduction of the kidney outer medulla, NOX4 mRNA was reduced by 77%, while NOX1 and NOX2 mRNA was unaffected. In control TALs, ANG II stimulated O₂⁻ production by 96%. In TALs transduced with NOX4 shRNA, ANG II-stimulated O₂⁻ production was not significantly different from the baseline. We concluded that NOX4 is the main catalytic isoform of NADPH oxidase that contributes to ANG II-stimulated O₂⁻ production by TALs.