The pulmonary vasculature is of great physiological/pathological significance. We have isolated and cultured microvessel endothelial cells (HuLEC) from lung tissue obtained from lung transplant recipients by modification of published methods. Pure cultures of HuLEC were isolated by mechanical disaggregation of the tissue prior to sequential dispase and trypsin digestion to obtain microvessel fragments. Magnetic beads (Dynabeads) coated with Ulex europaeus agglutinin-1 were then used to enhance the purity of cultures at the first passage. HuLEC formed contact-inhibited "cobblestone" monolayers on gelatin and fibronectin substrates and capillary-like "tubes" on Matrigel and accumulated acetylated low-density lipoprotein. Immunofluorescent characterization of these cells revealed the presence of von Willebrand Factor, angiotensin-converting enzyme, and thrombomodulin and the expression of antigens for the endothelial cell-specific monoclonal antibodies EN4, PAL-E, and H4-7/33. The endothelial origin of these cells was confirmed by the demonstration of the cell adhesion molecules, platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), and E-selectin (endothelial leukocyte adhesion molecule-1/ELAM-1) upon stimulation with TNF alpha. These cells should provide a useful tool for studying various aspects of pathology and biology of the pulmonary microvasculature in vitro.