Endothelial cells are indispensable components of the vascular system, and play pivotal roles during development and in health and disease. Their properties have been studied extensively by in vivo analysis of genetically modified mice. However, further analysis of the molecular and cellular phenotypes of endothelial cells and their heterogeneity at various developmental stages, in vascular beds and in various organs has often been hampered by difficulties in culturing mouse endothelial cells. In order to overcome these difficulties, we developed a new transgenic mouse line expressing the SV40 tsA58 large T antigen (tsA58T Ag) under the control of a binary expression system based on Cre/loxP recombination. tsA58T Ag-positive endothelial cells in primary cultures of a variety of organs proliferate continuously at 33 degrees C without undergoing cell senescence. The resulting cell population consists of blood vascular and lymphatic endothelial cells, which could be separated by immunosorting. Even when cultured for two months, the cells maintained endothelial cell properties, as assessed by expression of endothelium-specific markers and intracellular signaling through the vascular endothelial growth factor receptors VEGFR-2 and VEGFR-3, as well as their physiological characteristics. In addition, lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1) expression in liver sinusoidal endothelial cells in vivo was retained in vitro, suggesting that an organ-specific endothelial characteristic was maintained. These results show that our transgenic cell culture system is useful for culturing murine endothelial cells, and will provide an accessible method and applications for studying endothelial cell biology.