Incubation of bovine pulmonary microvascular endothelial (BPMVE) cells in low O2 content (95% N2-5% CO2) for 4 h increased monolayer permeability to dextran almost twofold and also increased the incidence of intercellular gaps and intracellular actin stress fibers. Hypoxic incubation decreased the extracellular matrix contents of fibronectin and vitronectin, proteins that serve as anchorage points for the endothelial cells. This state was reversed after 24 h of hypoxic incubation, and the BPMVE monolayer permeability to dextran was less than that of normoxic controls. The monolayer had fewer intercellular gaps and stress fibers, and the extracellular matrix contained increased amounts of fibronectin, vitronectin, and type I collagen. These alterations stimulated by 24 h of hypoxic incubation were resolved within 4 h of reoxygenation in room air supplemented with 5% CO2. These studies indicate that incubation of endothelial monolayers in hypoxic conditions first increases and then decreases monolayer permeability, through increased and decreased formation of intercellular gaps.