Mediators of angiogenesis such as VEGFs and angiopoietins may regulate pulmonary vascular permeability under both normal and pathologic conditions. Ephrin family receptor tyrosine kinases are expressed in the vasculature and also regulate angiogenesis under some circumstances but whether they too modulate lung vascular permeability is unknown. We hypothesized that stimulation of lung endothelial EphA receptors with ephrin-a1 ligand would alter pulmonary vascular permeability and tested this idea both in vivo and in vitro. We found that ephrin-a1 ligand and EphA2 receptors are expressed in distal normal lung vasculature and their expression is increased in injured lung, suggesting a link to mechanisms of increased permeability. Intravenous injection of ephrin-a1 caused a large increase in the leakage of labeled albumin into the lungs of rats within 30 minutes (293 ± 27 ng/mg dry lung vs. 150 ± 6, p < 0.01), accompanied by histologic evidence of the formation of endothelial disruptions. In cultured lung vascular endothelial cells, stimulation with ephrin-a1 increased monolayer permeability by 44% (p < 0.01), a similar permeability change as that seen with VEGF stimulation of the same cells. Ephrin-a1 stimulation both in vivo and in vitro was associated with histologic evidence for disruptions of tight and adherens junctions. These observations describe a novel role for ephrin-a1 and EphA receptors in the regulation of vascular permeability in the lung.