Introduction: The interaction between the epithelial and the microvascular endothelial cells during the oxidative stress of neutrophil-mediated injury is difficult to study. We have developed a novel in vitro pulmonary bilayer comprised of pulmonary epithelial cells with type II characteristics (A549) and isolated immortalized human pulmonary microvascular endothelial cells (HULEC). Methods: Using invertible transwell inserts (Costar), bilayers were constructed allowing appropriate physiologic orientation of the epithelial and endothelial cell. Bilayers were characterized in terms of viability, cell identification, integrity, and function. Results: Viability of A549 or HULEC cells on the bilayer was 94.1 ±2.9% and 94.8 ±3.4%, respectively, when compared to either A549 cells (93.3 ±3.3%), or HULEC cells (95 ±1.3%) in monolayer (p=NS). A549 cells selectively stained for lectin (97%) as compared to HULEC cells (0%). The endothelial isoform of nitric oxide synthase (eNOS) identified the HULEC cells (91 ±6% staining positively vs. 0% A549). The model revealed 100% of A549 and 92% of HULEC remained on their appropriate side of the membrane during culture. Bilayer resistance was similar to monolayers of A549 cells but higher than HULEC monolayers (p<0.01). The epithelial side demonstrated basal and induced surfactant secretion similar to monolayers of A549. Conclusions: This study provide baseline characteristics of a novel in vitro pulmonary bilayer. The cells are grown in the physiologic basolateral:basolateral orientation, which is important for neutrophil interactions and cellular functions. This data enables further study of each cell's contribution to inflammatory injury in the microcirculation of the lung.