In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.