Reactive oxygen species (ROS) produced by NADPH oxidases play important roles in the development of left ventricular hypertrophy (LVH) and remodelling. We recently demonstrated that the absence of the NADPH oxidase isoform, Nox2, reduced angiotensin II (AngII)-induced cardiac fibrosis (Johar et al. (2006)). In the heart, Nox2 is expressed mainly in endothelial cells and cardiomyocytes. Here, we investigated the role of endothelial Nox2 in cardiac remodelling in vivo. Male transgenic mice with endothelial-specific Nox2 overexpression (TG), previously generated in our laboratory, were compared to matched wild-type littermates (WT). AngII (1.1mg/kg/day) or saline were infused by osmotic minipump, implanted subcutaneously under 2% isoflurane anaesthesia, and the effects on cardiac structure and function assessed. Data were compared by 1-way ANOVA followed by Bonferroni’s multiple comparison test. Basal blood pressure and LV NADPH oxidase activity (lucigenin chemiluminescence) were similar in WT and TG mice. AngII infusion over 14 days induced similar hypertension in WT and TG mice, assessed by tail-cuff plesythmography and telemetry (170.2±11.6 vs. 170.4±12.3mmHg; n=10). LV NADPH oxidase activity was significantly higher in AngII-infused TG versus WT (by 27%; n=6, P<0.05). Both groups developed a similar degree of LVH: LV/body weight ratio, WT 4.8±0.2 vs. TG 4.7±0.2 mg/g; increase in echocardiographic septal thickness of 34% WT vs. 37% TG (n=10). Systolic function, assessed by LV pressure-volume analyses, did not differ significantly in WT vs. TG AngII-infused groups (ESPVR 8.9±0.7 in WT vs. 8.9±1.3 in TG; n=10). However, TG mice developed significant diastolic LV dysfunction as manifest by an increase in the LV end-diastolic pressure-volume relationship (EDPVR; TG: 0.2±0.0 vs. 0.5±0.1 after AngII, P<0.01; WT 0.2±0.0 vs. 0.3±0.0 after AngII; P=NS; n=10). Consistent with the increase in diastolic stiffness, AngII-treated TG hearts showed substantially higher interstitial cardiac fibrosis (Sirius red staining) than AngII-treated WT: 2.3±0.5 vs 1.3±0.1 %; n=6 P<0.05. These data suggest that endothelial Nox2 has a major role in the development of AngII-induced cardiac fibrosis independent of myocyte hypertrophy and hypertension. This contributes to increased diastolic stiffness and diastolic dysfunction.