Alveolar growth abnormalities and severe respiratory dysfunction are often fatal. Identifying mechanisms that control epithelial proliferation and enlarged, poorly septated airspaces is essential in developing new therapies for lung disease. The membrane-bound ligand ephrin-B2 is strongly expressed in lung epithelium, and yet in contrast to its known requirement for arteriogenesis, considerably less is known regarding the function of this protein in the epithelium. We hypothesize that the vascular mediator ephrin-B2 governs alveolar growth and mechanics beyond the confines of the endothelium. We used the in vivo manipulation of ephrin-B2 reverse signaling to determine the role of this vascular mediator in the pulmonary epithelium and distal lung mechanics. We determined that the ephrin-B2 gene (EfnB2) is strongly expressed in alveolar Type 2 cells throughout development and into adulthood. The role of ephrin-B2 reverse signaling in the lung was assessed in Efnb2LacZ/6YFDV mutants that coexpress the intracellular truncated ephrin-B2-b-galactosidase fusion and an intracellular point mutant ephrin-B2 protein that is unable to become tyrosine-phosphorylated or to interact with either the SH2 or PDZ domain-containing downstream signaling proteins. In these viable mice, we observed pulmonary hypoplasia and altered pulmonary mechanics, as evidenced by a marked reduction in lung compliance. Associated with the reduction in lung compliance was a significant increase in insoluble fibronectin (FN) basement membrane matrix assembly with FN deposition, and a corresponding increase in the a5 integrin receptor required for FN fibrillogenesis. These experiments indicate that ephrin-B2 reverse signaling mediates distal alveolar formation, fibrillogenesis, and pulmonary compliance.Keywords: arterial; fibronectin; a5b1 integrin; alveoli; pulmonary mechanics Lung development in mammalian species occurs through a series of overlapping stages, distinguished in terms of their histological appearance, and specifically the growth and differentiation of pulmonary epithelial structures. Although the proximal airways are completely formed by the end of gestation, the distal airways and alveoli continue their growth and maturation beyond the time of birth. Concurrent to the development of the epithelial structures, the pulmonary vasculature develops in a sequential fashion, via the coordinated processes of angiogenesis and vasculogenesis (1). Because of the close proximity of the developing airways and vasculature in the lung, vascular mediators have been suggested to influence pulmonary development. For instance, Chen and colleagues (2) and Schwarz and colleagues (3-5) used a mouse fetal lung allograft model to show that endothelial-monocyte activating polypeptide II can not only inhibit neovascularization, but can also significantly impair epithelial morphogenesis. Based on these and similar findings, our laboratory has focused on the role of vascular mediators during pulmonary development.Our studies have focused on the ephrin-B2 (EfnB2)...