NADPH oxidases (Noxs) are reactive oxygen species (ROS)-generating enzymes that exist in 7 distinct isoforms (Nox1-5, Duox1-2), each based on a different catalytic subunit. The prototypic Nox, i.e. Nox2, is involved in neutrophil microbicidal activity but in non-phagocytic cells, Nox2 and other Noxs generate low levels of ROS that modulate intracellular signalling. Nox2 and Nox4 are co-expressed in many cardiovascular cells, including endothelial cells and cardiomyocytes. Whereas Nox2 requires stimulation by agonists (eg, angiotensin II) and association with regulatory subunits for its activation, Nox4 is constitutively active and regulated mainly by its expression level. In studies in Nox2 knockout mice, we previously found that this isoform is involved in the development of cardiac hypertrophy, fibrosis and contractile dysfunction induced by angiotensin II, pressure overload or myocardial infarction. We have now generated models of cell-specific overexpression of Nox4 and Nox2 in order to define their cell- and isoform-specific roles. Endothelial-specific Nox4 overexpression resulted in enhanced endothelium-dependent vasodilatation which was attributable to H2O2-dependent hyperpolarization, rather than a change in nitric oxide bioactivity, and was associated with a reduced blood pressure. These mice also had blunted hypertensive responses to angiotensin II. In marked contrast, transgenic mice with endothelial-targeted Nox2 overexpression developed endothelial dysfunction and displayed increased hypertensive responses to angiotensin II. Cardiomyocyte-targeted Nox2 transgenic mice developed exaggerated cardiac hypertrophy following chronic pressure overload induced by aortic banding. In marked contrast, two independent lines of cardiomyocyte-targeted Nox4 transgenic mice were protected against pressure-overload induced cardiac hypertrophy, dilatation and contractile dysfunction. These findings indicate that Nox2 and Nox4 have dramatically different cardiovascular effects, even when expressed in the same cell type.