Dendrimers are hyperbranched synthetic well-defined molecules with a number of potential applications, especially in relation to the need for new antiviral agents. One subclass of dendrimers are peptide-derivatized dendrimers which consist of a peptidyl branching core and covalently attached surface peptide functional units. Few studies have addressed the potential uses of peptide dendrimers as direct-acting antiviral agents. Here, we report on the ability of two peptide dendrimers, SB105 and SB105_A10, to directly and almost completely inhibit human cytomegalovirus (HCMV) replication in both primary fibroblasts and endothelial cells; the agents were also found to inhibit murine CMV replication, whereas they were not able to inhibit adenovirus or vesicular stomatitis virus. The peptide dendrimers prevented adsorption of the HCMV to cells at 4 degrees C, whereas SB104, a dendrimer with a different amino acid sequence within the functional group and minimal anticytomegaloviral activity, was ineffective in blocking HCMV attachment. In effect, SB105_A10 bound to human cells through an interaction with cell surface heparan sulfate and thereby blocked virion attachment to target cells. These results indicate that the SB105 and SB105_A10 dendrimers could provide a useful starting point for the development of novel molecules to block HCMV infection.