Human cytomegalovirus (HCMV) strains were investigated to identify those with altered tropism for endothelial cells. In viral replication kinetics analysis, HCMV strain Toledo replicated poorly in aortic endothelial cells (AECs), and the virus count was reduced by 2-3 log units, in comparison with strain AD169. Virus entry at the cell surface for each strain was equivalent. However, immunofluorescence studies revealed a lack of immediate early viral antigen 72 expression, and direct blot hybridization failed to detect viral genomes in the nucleus of Toledo-infected AECs. Complementation assays were done to determine whether endothelial cell infectivity was dependent on a virus-encoded tropism factor. Pseudotype virus in endothelial cell cultures indicated that AD169 could provide trans factors to rescue Toledo during infection of endothelial cells. Collectively, these results show that a viral function provides an endothelial cell tropism factor for HCMV and plays a role during postentry infection events.