H oxidase (Nox)2 and Nox4 are the isoforms of Nox expressed in the macula densa (MD). MD-derived superoxide (O 2 Ϫ ), primarily generated by Nox2, is enhanced by acute ANG II stimulation. However, the effects of chronic elevations in ANG II during ANG II-induced hypertension on MD-derived O 2 Ϫ are unknown. We infused a slow pressor dose of ANG II (600 ng·min Ϫ1 ·kg Ϫ1 ) for 2 wk in C57BL/6 mice and found that mean arterial pressure was elevated by 22.3 Ϯ 3.4 mmHg (P Ͻ 0.01). We measured O 2 Ϫ generation in isolated and perfused MDs and found that O 2 Ϫ generation by the MD was increased from 9.4 Ϯ 0.9 U/min in control mice to 34.7 Ϯ 1.8 U/min in ANG II-induced hypertensive mice (P Ͻ 0.01). We stimulated MMDD1 cells, a MD-like cell line, with ANG II and found that O 2 Ϫ generation increased from 921 Ϯ 91 to 3,687 Ϯ 183 U·min Ϫ1 ·10 5 cells Ϫ1 , which was inhibited with apocynin, oxypurinol, or NS-398 by 46%, 14%, and 12%, respectively. We isolated MD cells using laser capture microdissection and measured mRNA levels of Nox. Nox2 and Nox4 levels increased by 3.7 Ϯ 0.17-and 2.6 Ϯ 0.15-fold in ANG II-infused mice compared with control mice. In MMDD1 cells treated with Nox2 or Nox4 small interfering (si)RNAs, ANG II-stimulated O 2 Ϫ generation was blunted by 50% and 41%, respectively. In cells treated with p22 phox siRNA, ANG II-stimulated O 2 Ϫ generation was completely blocked. In conclusion, we found that a subpressor dose of ANG II enhances O 2 Ϫ generation in the MD and that the sources of this O 2 Ϫ are primarily Nox2 and Nox4.hypertension; superoxide; tubuloglomerular feedback; kidney; angiotensin II KIDNEYS ARE VITAL in maintaining adequate extracellular fluid volume, and they do so by regulating the excretion of Na ϩ and water. Abnormalities in Na ϩ and water excretion may result in inappropriate salt and water retention, which can facilitate hypertension. One of the mechanisms that the kidney possesses to regulate salt excretion is via the macula densa (MD). MD cells are modified epithelial cells located at the distal segment of the thick ascending limb (TAL) that comprise an integral component of the juxtaglomerular apparatus (JGA). MD cells serve as luminal sensors of NaCl concentration; they sense increase in NaCl delivery, to which they respond by initiating a sequence of events that results in an increase in adenosine and/or ATP release. These, in turn, constrict the afferent arterioles, lower capillary hydraulic pressure, decrease the single nephron glomerular filtration rate (SNGFR), and decrease tubular flow back to normal levels. This series of events is collectively termed as the tubuloglomerular feedback (TGF) response. The TGF response establishes a negative feedback by which a change in tubular flow to the MD induces a reciprocal change in SNGFR, thus preventing acute fluctuations of flow and NaCl delivery in the distal nephron (7,42,51,53,59,60). Hence, TGF limits urinary volume and Na ϩ excretion and thus may contribute to the maintainance of salt and water homeostasis. Any abnormality in th...