Lymphatic endothelial cell (LEC) culture is associated with several problems. There are ethical concerns about the collection of LECs from humans, in addition to the concern that LECs from different individuals might exhibit variable behavior. Properties of LECs such as morphology can also change when they are cultured for prolonged periods. These problems may hinder the analysis of LEC properties and functions, and obstruct elucidation of mechanisms underlying lymphatic system-mediated cancer metastasis. To overcome these problems, we increased the culture duration of an established LEC line by generating a LEC line stably expressing high levels of the large T antigen of simian virus 40 (LEC-SV). This LEC-SV line could be cultured for approximately twice as long as the parental LEC line. LECs are thought to be involved in hormone-dependent lymphogenous metastasis; therefore, the response of LEC and LEC-SVs to estrogen stimulation was also investigated. Levels of mRNA for three LEC marker genes, Flt-4 , Xlkd-1 , and Prox1 , were significantly higher in β-estradiol-treated parental LECs and LEC-SVs compared to vehicle-treated LECs and LEC-SVs. This LEC-SV line should be a valuable tool for analyzing the properties and functions of lymphatic vessels and endothelial cells.