Endothelial barrier dysfunction is an important contributor to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Even though approaches that target the prevention and repair of endothelial barrier dysfunction are clearly needed, our understanding of the molecular regulation of pulmonary microvascular endothelial permeability remains incomplete. Cultured pulmonary microvascular endothelial cells represent an attractive paradigm for the study of barrier function. Here, we describe a method for the harvest, identification and culture of human lung microvascular endothelial cells (HLMVEC). HLMVEC thus obtained, grow as a monolayer, exhibit contact inhibition and have the typical cobblestone appearance. They express endothelial proteins, such as von Willebrand factor and endothelial nitric oxide synthase and take up an acetylated LDL. Furthermore, HLMVEC respond predictably and with superior sensitivity to the barrier disruptive effects of Gram positive and Gram negative bacterial products, thrombin, vascular endothelial growth factor and microtubule disrupting agents. These HLMVEC present an in-house-derived alternative to commercially available human cells for the study of mechanisms contributing to ALI and ARDS.