Reactive oxygen species (ROS) signal vital physiologic processes including cell growth, angiogenesis, contraction and relaxation of vascular smooth muscle. Because Cyp4/20-HETE has been reported to enhance angiogenesis, pulmonary vascular tone, and eNOS function, we explored the potential of this system to stimulate bovine pulmonary endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time- and concentration dependent manner, as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate (DCF). An analog of 20-HETE elicits no increase in ROS, and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE induced ROS production in BPAECs is blunted by pretreatment with PEG-SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47phox association with gp91. These data support 20-HETE stimulated, NADPH oxidase derived and Rac1/2 dependent ROS production in BPAECs. 20-HETE promotes translocation of p47^phox and tyrosine phosphorylation of p47^phox in a time- dependent manner, as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through by activation of NADPH oxidase within minutes of application of the lipid.