Microvascular endothelial cells play a critical role in tumor progression and metastasis by forming capillary networks that encourage tumor growth and by promoting the attachment of circulating tumor cells to the vascular wall of distant tissues. Efforts to study the molecular mechanisms that mediate these complex processes in different anatomical compartments have been impeded by difficulties in the isolation and propagation of endothelial cells from different organs. To overcome these limitations, we used two-color flow cytometry to identify and select microvascular endothelial cells from primary cultures obtained from different organs of mice whose tissues harbor a temperature-sensitive SV40 large T antigen (H-2K^b-tsA58 mice; ImmortoMice). The selection strategy targeted cell populations expressing the inducible endothelial cell adhesion molecules, E-selectin and VCAM-1, and proved successful in generating microvascular endothelial cell lines from a number of different organs. When cultured under permissive temperatures (33°C), individual cell lines displayed doubling times consistent with endothelial cells possessing an angiogenic phenotype. The transfer of endothelial cells to nonpermissive temperatures (37°C) resulted in cell differentiation and the induction of a quiescent state. Established cell lines exhibited several inherent endothelial properties, including the expression of constitutive and inducible levels of endothelial cell adhesion molecules E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, internalization of acetylated low-density lipoprotein, and formation of loop structures on Matrigel surfaces. The immortalized endothelial cell lines established from H-2K^b-tsA58 mice provide, for the first time, a cell culture system to examine factors regulating angiogenesis and tumor cell arrest in different organ systems.