We focused on the role of membrane bound sugar residues in the infection of fibroblasts and monocyte-like cells with human cytomegalovirus (HCMV). Treatment of phorbol 12-myristate 13-acetate (PMA) differentiated monocyte-like cells THP-1 or human fibroblasts MRC-5 with lectins specific for N-acetylneuraminic acid (NeuAc) blocked infection with HCMV. HCMV failed to infect sialidase-treated differentiated THP-1 cells or MRC-5 cells. By using NeuAc, N-glycolylneuraminic acid (NeuGl) and alpha 2-3, but not alpha 2-6, sialyl-oligosaccharide, the infection of cells was less efficient. NeuAc was more potent inhibitor than NeuGl. These observations suggest that the sialic acid specificity and the nature of the interglycosidic linkage at the end of the complex carbohydrates may play an important role. Analogous experiments indicated that HCMV binds to N-acetylglucosamine (GlcNAc) in addition to NeuAc. Human cytomegalovirus infection in differentiated THP-1 cells and in human fibroblasts was inhibited by incubation of the virus with 20 micrograms/ml of heparin before and during the adsorption period. Treatment of the cells with heparinase or heparitinase inhibited infection with HCMV. We emphasized the role of NeuAc and GlcNAc and heparan sulfate proteoglycans at the surface of the cells, in the early steps of infection of both human fibroblasts and PMA differentiated monocyte-like cells with HCMV.