Heparan sulfate moieties serve as receptors for initial binding of herpes simplex virus types 1 and 2 (HSV-1 and -2) to cells. Deletion of HSV-1 glycoprotein C (gC-1) but not HSV-2 gC (gC-2) results in virions with reduced specific binding activity (virus particles bound per cell) and specific infectivity (p.f.u. per particle), suggesting that for HSV-1, but not HSV-2, gC plays a major role in mediating virus attachment. To test the hypothesis that glycoprotein B (gB), the other heparin-binding glycoprotein, mediates HSV-2 attachment, HSV-2 viruses deleted in gB-2 alone or deleted in both gB-2 and gC-2 were constructed. These viruses were grown on complementing or non-complementing cells and were compared with parental HSV-2(G) or a gC-2-deleted HSV-2 mutant (with respect to ability to bind and infect cells). At equivalent input concentrations of purified virions, significantly fewer gB-2-deleted virions bound to cells compared to parental HSV-2(G) or virus grown on complementing cells. In addition, viruses deleted in gB-2 were non-infectious. No immediate early proteins were detected in cells infected with gB-2-deleted virus harvested from non-complementing Vero cells, whereas these proteins were readily detected 4 h post-infection in cells infected with virus grown on complementing cells or with parental viruses. Viruses deleted in gB-2 failed to spread cell to cell, as evidenced by the inability to form plaques. Together these studies demonstrate that gB-2 plays a key role in mediating HSV-2 attachment and is required for entry and cell-to-cell spread. This glycoprotein is an important target for development of novel antiviral drugs.