Endothelial cells (EC) are common targets of permissive infection by human cytomegalovirus (HCMV) in vivo during acute disease. However, studies of HCMV-EC interactions in vitro have generated discordant results. While lytic infection of cultured venous EC has been well established, Fish et al. (K. N. Fish, C. Soderberg Naucler, L. K. Mills, S. Stenglein, and J. A. Nelson, J. Virol. 72:5661–5668) have reported noncytopathic persistence of the virus in cultured aortic EC. We propose that interstrain differences in viral host cell tropism rather than the vascular bed of origin of infected EC might account for these discrepancies. In the present investigation we compared the responses of EC derived from human adult iliac artery, placental microvasculature, and umbilical vein to infection with various HCMV strains. Regardless of the vascular bed of origin, infection with EC-propagated HCMV strains induced 100% efficient cytopathic change progressing to complete lysis of inoculated monolayers. While fibroblast-propagated strains persisted at low titer in infected arterial EC cultures, they were also cytolytic for individual infected cells. The finding of cytopathic lytic infection of arterial EC by HCMV implicates a mechanism of vascular injury in the pathogenesis of HCMV infection.