CD40 has been identified as an important molecule for a number of processes, such as immune responses, inflammation, and the activation of endothelia. We investigated CD40 in endothelial cells (EC) following infection with an endotheliotropic strain of human cytomegalovirus (HCMV). Between 8 and 72 h postinfection, we observed a significant increase in CD40 levels on the surface of infected EC, as measured by fluorescence-activated cell sorting analysis. As a consequence of CD40 upregulation, increased levels of E-selectin were found on infected EC after stimulation with CD154-expressing T cells. Enhanced expression of CD40 was specific for EC, since infection of fibroblasts did not result in the upregulation of CD40. The addition of neutralizing antibodies as well as UV inactivation of virus completely prevented the upregulation of CD40 on EC. Also, laboratory-adapted HCMV strain AD169 was not able to induce CD40 on EC. De novo protein synthesis was necessary for the increased surface expression. At early times (4 to 24 h) postinfection, this change was not accompanied by increased levels of CD40 protein or mRNA. At late times (48 to 96 h) postinfection, increased amounts of CD40 protein and mRNA were detected. Immunohistochemical analysis of infected tissues demonstrated elevated levels of CD40 on HCMV-infected EC in vivo. Thus, infection of EC by HCMV may result in the activation of endothelia and in the augmentation of inflammatory processes.