Nox2-containing NADPH oxidases are reported to be involved in the development of cardiac fibrosis in response to chronic angiotensin II infusion, but the cellular source(s) of Nox2 involved in fibrosis remains unclear. We investigated the role of endothelial Nox2 in angiotensin II-induced left ventricular hypertrophy (LVH). Male transgenic mice with endothelial-specific overexpression of Nox2 were compared with matched wild-type (wt) littermates after angiotensin II (1.1 mg/kg per day) or saline infusion for 14 days. Basal blood pressure and left ventricular NADPH oxidase activity were similar in wt and transgenic mice. After angiotensin II infusion, both wt and transgenic groups developed similar hypertension (170.2±11.6 vs 170.4±12.3 mm Hg; n=10) and hypertrophy (left ventricular/body weight ratio 4.8±0.2 vs 4.7±0.2 mg/g; and echocardiographic septal thickness increased by 34% wt and 37% transgenic mice; n>10). NADPH oxidase activity was higher in angiotensin II-infused transgenic compared with wt left ventricles (p<0.05). Interestingly, transgenic hearts showed significantly more interstitial cardiac fibrosis (2.3±0.5 vs 1.3±0.1% by Sirius red staining; p<0.05; n=6). In concurrence with a greater increase in inflammatory cell infiltration in transgenic left ventricles compared with wt (55% increase in CD45 staining; p<0.05; n=6). Left ventricular systolic function assessed in vivo by left ventricular pressure–volume analysis was similar in both angiotensin II groups (ESPVR, 8.9±0.7 in wt vs 8.9±1.3 mm Hg/μl in transgenic mice; p=ns; n=10), whereas diastolic stiffness increased significantly only in the angiotensin II transgenic group (EDPVR increased from 0.2±0.0 to 0.5±0.1 mm Hg/μl in transgenic mice; p<0.05; and from 0.2±0.0 to 0.3±0.0 mm Hg/μl in wt; n=10). These results indicate that endothelial-specific overexpression of Nox2 significantly enhances the development of angiotensin II-induced cardiac fibrosis and left ventricular diastolic dysfunction, independent of changes in hypertrophy.