HPMECs were successfully isolated by differential trypsinization from peripheral lung lobes. The cells proliferated rapidly in EGM-MV with 10% FBS and were serially cultivated for more than 20 passages (1:4 split ratio) in vitro. Cells were characterized as endothelial based upon their cobblestone morphology, the presence of factor VIII-related antigen, incorporation of DiI-Ac-LDL, tubule-like structure formation in Matrigel, and positive staining for ACE. Adhesion molecules were tested at passage 3 and passage 12. Cells demonstrated intense staining for PECAM-1 both unstimulated and stimulated with TNF-alpha (20 ng/ml). The adhesion molecules ICAM-1, VCAM-1, ELAM-1, and P-selectin differed in expression on unstimulated cells. ICAM-1 was constitutively expressed on unstimulated cells and the expression was increased by TNF-alpha stimulation (20 hr). In contrast, VCAM-1, ELAM-1, and P-selectin were not detected on unstimulated cells but were detected after stimulation with TNF-alpha. The inducibility of adhesion molecules was different. VCAM-1 (10 hr) and ELAM-1 (4 hr) were expressed more strongly than P-selectin (minutes to 4 hr). The adhesion molecule profile found on passage 12 was the same as on passage 3. CD36 was not detected on both unstimulated and stimulated (4 and 8 hr) cells. The peak of adhesion of HL-60 cells to TNF-alpha activated HPMEC monolayers was around 8 hr. The results indicate that HPMEC can be continuously grown in vitro for many passages without losing their adhesion molecule expression. This expression of adhesion molecules confirms that HPMECs might be a good in vitro model in the understanding of various aspects of pulmonary microvascular endothelial cell function and may be useful as the basis for studies of adhesion molecule targeted therapies of pulmonary inflammatory diseases.