Endothelial cells (EC) are an important cell type for human cytomegalovirus (CMV) pathogenesis. To characterize better the role of EC in primate CMV natural history, rhesus macaque microvascular EC (MVEC) were purified from fetal brain and analysed for infectivity by rhesus cytomegalovirus (RhCMV). Rhesus brain MVEC (BrMVEC) in culture were positive for von Willebrand factor and CD105 expression, uptake of acetylated low-density lipoprotein, and formation of capillary-like tubules on Matrigel, all phenotypic hallmarks of EC. BrMVEC were fully permissive for infection by RhCMV strain 68-1, and detectable plaques formed within 5 days of infection. Infectivity of BrMVEC by RhCMV could be reduced, but not abolished, by treatment of cells either before or during infection with pro-inflammatory mediators tumour necrosis factor-alpha, interleukin-1beta or phorbol 12-myristate 13-acetate. These results demonstrate that in vitro infection of rhesus BrMVEC is a dynamic process that is influenced by activation conditions.