Oxidative stress is implicated in the genesis of cardiovascular diseases through the inactivation of nitric oxide and modulation of redox-sensitive signalling pathways. A family of NADPH oxidases (Noxs) form an important source of reactive oxygen species (ROS) within the vessel wall, with the Nox2 and Nox4 isoforms being the major contributors to endothelial ROS. The involvement of Nox2 in endothelial dysfunction is well established, but the role of Nox4 remains uncertain with recent evidence indicating that it has distinct mechanisms of regulation to Nox2. The aim of the present study was to investigate the in vivo role of Nox4 in the endothelium. Transgenic mice (Tg) were generated with endothelial-targeted overexpression of Nox4 using a Tie2 promoter construct, and were backcrossed into a C57Bl6/j background. Tg mice had 2-fold greater Nox4 mRNA expression and 3-fold greater Nox4 protein in coronary microvascular endothelial cells (CMEC) compared with wild-type littermates (WT)(n=4; p<0.05). Tg CMEC had increased NADPH-dependent superoxide production (237.6±2.7 vs. 186.5±7.1 integrated RLU; n=3, p<0.01) and increased hydrogen peroxide generation (homovanillic acid assay) compared to WT (7.60±0.70 vs. 3.22±0.42 µM H₂O₂/10⁵ cells; n=3, p<0.01). No changes were noted in expression of p22phox, SOD1-3 or catalase mRNA. In vivo systolic and diastolic blood pressure measured by telemetry was significantly lower in TG mice compared to WT (systolic 117.4±1.9 vs. 125.5±2.1 mmHg and diastolic 90.1±2.0 vs. 98.1±2.1 mmHg; n=5, p<0.05). Isolated preconstricted aortic rings from TG mice revealed enhanced acetylcholine-induced vasorelaxation compared to WT (-log EC₅₀ 7.76±0.07 vs. 7.20±0.05; n=12, p<0.001), a difference that was abolished by catalase (1500 U/ml). Similarly, coronary microvascular resistance in isolated Langendorff-perfused hearts was reduced to a greater extent by acetylcholine in TG than WT (-log EC₅₀ 5.59 ± 0.27 vs.4.80 ± 0.48; n=5, p<0.05), an effect also abolished by catalase (1500 U/ml). Chronic 7-day administration of the SOD and catalase-mimetic, EUK-8, in vivo abolished the difference in blood pressure between TG and WT (Difference 11.7±1.6 mmHg pre- vs. 1.6±4.3 mmHg post-EUK-8). Taken together, these results indicate that overexpression of Nox4 within the endothelium has unforeseen beneficial effects on both vasomotor tone and blood pressure, probably through generation of hydrogen peroxide. These results suggest that endothelial Nox4 and Nox2 have distinct and contrasting functions in vivo .