The purposes of this study were to determine whether heparin would block human cytomegalovirus (HCMV) infection of skin fibroblast (SF) cells and to identify HCMV envelope glycoproteins which might have affinity for heparin. It was determined that soluble heparin in concentrations of 5 to 20 micrograms/ml was capable of blocking HCMV infection of SF cells. However, after virus had adsorbed to the SF cells, heparin lost its ability to block infection. It was also determined that treatment of SF cells with heparinase to remove cell surface heparinlike moieties prevented HCMV infection of SF cells. These data showed that HCMV, like other herpesviruses, adsorbed to cells by binding cell surface heparin. Heparin affinity chromatography was done to determine which HCMV envelope glycoproteins bound heparin. HCMV envelope glycoproteins were solubilized in a nonionic detergent and applied to a heparin affinity column. An HCMV glycoprotein complex designated gC-II was the major component to bind to immobilized heparin and elute in the presence of soluble heparin.