Primary cultures of bovine microvascular endothelial cells (BME) isolated from the adrenal cortex, are commonly used to study vascular endothelium, but have a limited life span. To circumvent these limitations, we have immortalized BME cells with either simian virus 40 (SV40) or with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have investigated whether the clonal populations obtained, maintain differentiated properties characteristic of microvascular endothelium. Immortalized cells were characterized for maintenance of typical endothelial morphology, marker expression, and functional characteristics including uptake of Acetylated low-density lipoprotein (Ac-LDL), capillary-like tube formation in three-dimensional collagen gels, as well as metalloproteinase (MMP) and plasminogen activator (PA)-mediated extracellular proteolysis. Whilst immortalization of BME cells with SV40 was associated with loss of endothelial-specific properties, hTERT-BME exhibited an endothelial phenotype similar to that of wild-type endothelial cells. Specifically, they showed a typical cobblestone morphology, were contact-inhibited, expressed endothelial cell-specific markers (e.g., CD31, vWF) and both fibroblast growth factor receptor 1 (FGFR-1) and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, they expressed receptors for LDL. Importantly, when grown on collagen gels, hTERT-BME cells underwent MMP-dependent tube-like structure formation in response to VEGFR-2 activation. In a collagen gel sandwich assay, hTERT-BME formed tubular structures in the absence of exogenously added angiogenic cytokines. Sustained tube formation was induced by VEGF-A alone or in combination with FGF-2. From 17 sub-clones that displayed a non-transformed phenotype, a high proliferative capacity and tubulogenic properties in three-dimensional collagen gels, we isolated two distinct subpopulations that display a highly specific response to VEGF-A or to FGF-2. We have generated hTERT-BME cells that maintain endothelial-specific properties and function and have isolated clones that respond differentially to VEGF-A or FGF-2. These immortalized cell lines will facilitate the study of endothelial cell biology.