Reactive oxygen species (ROS) control the integrity of the vascular endothelium. Our laboratory has recently shown that transduction of human umbilical vein endothelial cells (HUVECs) with an active variant of the small GTPase Rac promotes the production of ROS, ROS-dependent activation of p38 mitogen-activated protein kinase, and loss of vascular/endothelial-cadherin-mediated cell-cell adhesion. Here we show that HUVECs express mRNAs for NOX2 as well as NOX4 mRNA, but not for NOX1 or NOX3. Interestingly, NOX4 was expressed at 100-fold higher levels compared with NOX2. NOX4-green fluorescent protein largely localizes to an intracellular compartment that costained with a marker for the endoplasmic reticulum, and its distribution did not overlap with lysosomes, Weibel-Palade bodies, or mitochondria. The NOX2-regulatory proteins p47(phox) and p67(phox) associated with the actin cytoskeleton and were found in cell protrusions and membrane ruffles, colocalizing with Rac1. This translocation to the cell periphery was promoted by tumor necrosis factor (TNF)-alpha. Finally, scavenging of ROS was found to impair TNF-alpha-induced cytoskeletal rearrangements and the formation of a confluent endothelial monolayer. Together, these data prove the differential mRNA expression of NOX family members in human endothelium and indicate that these NOX proteins and their regulators may be involved in the control of endothelial cell spreading, motility, and cell-cell adhesion.