20-hydroxyeicosatetraenoic acid (20-HETE) is an endogenous cytochrome P450 product present in vascular smooth muscle and uniquely located in the vascular endothelium of pulmonary arteries (PAs). 20-HETE enhances reactive oxygen species (ROS) production of bovine pulmonary artery endothelial cells (BPAECs) in an NADPH oxidase dependent manner, and is postulated to promote angiogenesis via activation of this pathway in systemic vascular beds. We tested the capacity of 20-HETE or a stable analog of this compound 20-hydroxy-eicosa-5(Z),14(Z)-dienoic acid (20-5,14-HEDE) to enhance survival and protect against apoptosis in BPAECs stressed with serum starvation. 20-HETE produced a concentration dependent increase in numbers of starved BPAECs and increased BrDu incorporation. Caspase-3 activity, nuclear fragmentation studies, and MTT assays supported protection from apoptosis and enhanced survival of starved BPAECs treated with a single application of 20-HETE. Protection from apoptosis depended upon intact NADPH oxidase, PI3 kinase and ROS production. 20-HETE stimulated ROS generation by BPAECs was blocked by inhibition of PI3 kinase or Akt activity. These data suggest 20-HETE associated protection from apoptosis in BPAECs required activation of PI3 kinase, Akt, and generation of ROS. 20-HETE also protected against apoptosis in BPAECs stressed by lipopolysaccharide (LPS), and in mouse PAs exposed to hypoxia reoxygenation ex-vivo. In summary, 20-HETE may afford a survival advantage to BPAECs through activation of pro-survival PI3 kinase and Akt pathways, NADPH oxidase activation and NADPH oxidase derived superoxide.