Since the introduction of culture methods for umbilical vein endothelial cells, many successful attempts for culturing endothelial cells have been reported. The successful establishment of immortalized cell lines of organ specific endothelium, however, has been rare. Primary cultured endothelial cells isolated from the Copenhagen rat prostate were immortalized by infection with an Adenovirus-12 SV40 hybrid virus. Two immortal cell lines of Copenhagen rat prostate endothelium were established and designated YPEN-1 and YPEN-2. The established cell lines have continued to proliferate more than 80 population doublings and have not undergone senescence. They stain positively for but are nonproducers of SV4O0 T-antigen and grow in a monolayer with a cobblestone appearance. They demonstrate Dil-Ac-LDL uptake as an endothelial marker. YPEN-1 and YPEN-2 cells exhibit positive staining for endothelin and MRC OX-43 and express Integrin a6 beta 1 and Integrin beta 3 on their plasma membrane and demonstrate tube formation in Matrigel. Doubling times of YPEN-1 and YPEN-2 are 26 hours and 21 hours, respectively. Genetically, YPEN-1 and YPEN-2 are both diploid. These cell lines of rat prostate endothelial origin should be useful for studying angiogenesis and its inhibition.