A number of reports have suggested that human cytomegalovirus (HCMV)-infected fibroblasts are resistant to natural killer (NK) lysis, and that the HCMV-encoded human leucocyte antigen (HLA) class I homologue UL18 may be responsible for this effect. While fibroblasts are easy to infect in vitro, their role in HCMV pathogenesis in vivo is unclear. Here, we have established systems to address NK recognition of infected endothelial cells and macrophages, two important HCMV cellular reservoirs in vivo. The HCMV-infected endothelial cells exhibited increased resistance to NK killing, and, in most experiments, infected macrophages demonstrated a decreased susceptibility to NK lysis. Infection with the mutant HCMV strain RV670, lacking the genes US1-9 and US11 that are responsible for downregulation of HLA class I molecules, also led to decreased NK susceptibility. Furthermore, reduced NK susceptibility was independent of the expression of the HLA class I homologue UL18, since cells infected with the UL18Delta HCMV strain were also less susceptible to NK killing. These results suggest that HCMV-induced resistance to NK cytotoxicity in endothelial cells and macrophages is independent of known pathways that interfere with the expression of cellular HLA class I A, B and C surface antigens and the HCMV encoded class I homologue UL18.