We assessed the effect of varying levels of hyperoxia on14C-albumin flux across bovine pulmonary artery endothelial cell (BPAEC) monolayers. Endothelialized nitrocellulose filters were mounted in Ussing-type chambers which were filled with cell culture medium (M 199). Equimolar amounts of14C-labeled and unlabeled albumin were added to the“hot”and“cold”chambers, respectively, and the monolayers were exposed to 3 hours of varying levels of oxygen (16%, 30%, 40%, 60%, and 95%). When compared to 16% O2, exposure to hyperoxic gas mixtures of 40% or greater progressively increased albumin permeability across endothelial monolayers within 3 hours to a value 2.5 times higher at 95% O2compared to 16% O2(p<0.001). Hyperoxia-induced permeability increases were prevented by catalase, superoxide dismutase, desferriox-amine, and allopurinol. Our data indicate that hyperoxia induces endothelial permeability changes more rapidly than previously reported even at O2concentrations as low as 40%.