Angiotensin (ANG) II (AngII) and aldosterone contribute to the development of interstitial cardiac fibrosis. We investigated the potential role of a Nox2‐containing NADPH oxidase in aldosterone‐induced fibrosis and the involvement of this mechanism in AngII‐induced effects. Nox2^−/− mice were compared with matched wild‐type controls (WT). In WT mice, subcutaneous (s.c.) AngII (1.1 mg/kg/day for 2 wk) significantly increased NADPH oxidase activity, interstitial fibrosis (11.5±1.0% vs. 7.2±0.7%; P<0.05), expression of fibronectin, procollagen I, and connective tissue growth factor mRNA, MMP‐2 activity, and NF‐kB activation. These effects were all inhibited in Nox2^−/− hearts. The mineralocorticoid receptor antagonist spironolactone inhibited AngII‐induced increases in NADPH oxidase activity and the increase in interstitial fibrosis. In a model of mineralocorticoid‐dependent hypertension involving chronic aldosterone infusion (0.2 mg/kg/day) and a 1% Na Cl diet (“ALDO”), WT animals exhibited increased NADPH oxidase activity, pro‐fibrotic gene expression, MMP‐2 activity, NF‐kB activation, and significant interstitial cardiac fibrosis (12.0±1.7% with ALDO vs. 6.3±0.3% without; P<0.05). These effects were inhibited in Nox2^−/− ALDO mice (e.g., fibrosis 6.8±0.8% with ALDO vs. 5.8±1.0% without ALDO; P=NS). These results suggest that aldosterone‐dependent activation of a Nox2‐containing NADPH oxidase contributes to the profibrotic effect of AngII in the heart as well as the fibrosis seen in mineralocorticoid‐dependent hypertension.—Johar, S., Cave, A. C., Narayanapanicker, A., Grieve, D. J., Shah, A. M. Aldosterone mediates angiotensin II‐induced interstitial cardiac fibrosis via a Nox2‐containing NADPH oxidase. FASEB J. 20, E846–E854 (2006)