Glomerular endothelial cells (GEnC) are specialized cells with important roles in physiological filtration and glomerular disease. Despite their unique features, GEnC have been little studied because of difficulty in maintaining them in cell culture. We have addressed this problem by generation of conditionally immortalized (ci) human GEnC using technology with which we have previously produced ci podocytes. Primary culture GEnC were transduced with temperature-sensitive simian virus 40 large tumour antigen and telomerase using retroviral vectors. Cells were selected, cloned, and then characterized by light and electron microscopy (EM), response to vascular endothelial growth factor (VEGF), and tumour necrosis factor (TNF)alpha, expression of endothelial markers by focused gene array, immunofluorescence and Western blotting, and formation and behaviour of monolayers. CiGEnC proliferated at the permissive temperature (33 degrees C) and became growth arrested at the non-permissive temperature (37 degrees C). CiGEnC retained morphological features of early-passage primary culture GEnC up to at least p41, confirming successful immortalization. EM demonstrated fenestrations, increased in number by VEGF. mRNA analysis confirmed expression of the endothelial markers platelet endothelial cell adhesion molecule 1, intercellular adhesion molecule 2, VEGF receptor 2, and von Willebrand factor, validated by immunofluorescence and Western blotting. CiGEnC also expressed Tie2, and TNFalpha upregulated E-selectin. CiGEnC formed monolayers with barrier properties responsive to cyclic adenosine 3',5' monophosphate (cAMP) and thrombin. CiGEnC retain the markers and behaviour of primary culture GEnC. They express fenestrations which are upregulated in response to VEGF. These cells are a unique resource for further study of GEnC and their roles in glomerular filtration, glomerular disease, and response to glomerular injury.