A rapid, reproducible method for the isolation of murine endothelial cells (ECs) has been developed. Murine ECs were highly enriched by collagenase digestion of mechanically minced lung and subcutaneous sponge implants followed by specific selection with rat anti-mouse CD31 (i.e., PECAM-1) monoclonal antibody-coated magnetic beads (Dynabeads). Pure EC populations were isolated from primary cultures by a second cycle of immunomagnetic selection. The cells from the lung were then cloned by a limiting-dilution method to exclude the possibility of nonendothelial cell contamination. Of the 300 cells plated, 29 clones (approximately 10%) were obtained. The clones were positive for CD31 as measured by flow cytometry, and one clone from the lungs (1G11) and the cells from sponge implants (designated as SIECs) were then subjected to subsequent culture in vitro for 40 and 30 passages (up to 5 months), respectively. Characterization was performed on cells between passage 3 and 10. Both cell types formed contact-inhibited monolayers on gelatin and capillary-like "tubes" on Matrigel. However, 1G11 cells exhibited a "cobblestone" morphology, whereas SIECs had a fibroblast-like appearance at confluence. By flow cytometry and enzyme-linked immunosorbent assay, these cells constitutively expressed CD31, VE-cadherin (cadherin-5), CD34, ICAM-1, VCAM-1, and P-selectin. After stimulation with 30 ng/mL of tumor necrosis factor-alpha, the cells became positive for E-selectin (at 4 hours poststimulation) and the expression of ICAM-1, VCAM-1, and P-selectin was upregulated (after 24 hours of stimulation). The presence of VE-cadherin in 1G11 cells and SIECs was confirmed by fluorescence microscopy and Northern blot analysis. The phenotype and morphology of both cell types were stable during 5 months of culture, and there was no evidence of overgrowth by contaminating cells. Taken together, the approach outlined herein may provide a general strategy for the isolation and culture of ECs from a variety of murine tissues. The general strategy outlined here is simple, effective, and flexible, allowing the inclusion of further positive or negative selection steps.