The pulmonary endothelium is a single-cell layer forming the inner lining of a vast network of arteries, veins, and capillaries in the lung. Its main function is to regulate the contractility of underlying vascular smooth muscle cells (SMCs), which determines vascular tone and allows adaptation of blood flow to oxygenative conditions. Low oxygen tension (hypoxia) causes vasoconstriction of pulmonary vasculature and, depending on the duration of hypoxia, this effect may be reversed by reoxygenation. The key role of the pulmonary endothelium in the regulation of vascular tone has focused considerable attention on the effects of hypoxia/reoxygenation on pulmonary endothelial barrier function. Hypoxia increases endothelial permeability, which is believed to promote vasoconstriction by facilitating the leakage of vasoactive agents from the blood to the underlying SMCs. Data show that Rho GTPases RhoA and Rac1 regulate pulmonary endothelial barrier function in response to changes in oxygen tension. This chapter describes methods to isolate and culture primary pulmonary artery endothelial cells, to measure changes in endothelial barrier function and reactive oxygen species production, and to study the role of Rho GTPases in endothelial responses to hypoxia and reoxygenation.