Reactive oxygen species (ROS) have been shown to promote endothelial proliferation. A NOX2-containing NADPH oxidase generates ROS in leukocytes, but also in endothelial cells (EC). However, the contribution of newly identified homologues of NOX2 to endothelial ROS generation and proliferation is unclear. We compared the role of NOX1, NOX4 and NOX5 to NOX2 in these endothelial responses. NOX2, NOX4 and NOX5 were expressed at the mRNA and protein levels in EC whereas NOX1 expression was barely detectable. All proteins colocalized with markers of the endoplasmic reticulum (ER). In addition, NOX2, but not the other NOX proteins, also colocalized with actin at the plasma membrane. Bimolecular fluorescent complementation showed an interaction between the NOX proteins and p22phox. Whereas NOX2, NOX4 and NOX5 modulated ROS production and proliferation as was shown using expression vectors and specific siRNA, NOX1 did not significantly contribute to these responses. These data show that EC express NOX1, NOX2, NOX4 and NOX5 in the ER where they interact with p22phox. NOX2, NOX4 and NOX5, but not NOX1 contribute to basal endothelial ROS generation and proliferation. These findings indicate that a complex relation between several NOX homologues controls endothelial function and redox signalling.