Variants of herpes simplex virus type 2 (HSV-2) generated by virus passage in GMK-AH1 cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to PI-88 in their initial infection of cells and/or their cell-to-cell spread. The major alteration detected in all variants resistant to PI-88 in the initial infection of cells was a frameshift mutation(s) in the glycoprotein G (gG) gene that resulted in the lack of protein expression. Molecular transfer of the altered gG gene into the wild-type background confirmed that the gG-deficient recombinants were resistant to PI-88. In addition to PI-88, all gG-deficient variants of HSV-2 were resistant to the sulfated polysaccharide heparin. The gG-deficient virions were capable of attaching to cells, and this activity was relatively resistant to PI-88. In addition to having a drug-resistant phenotype, the gG-deficient variants were inefficiently released from infected cells. Purified gG bound to heparin and showed the cell-binding activity which was inhibited by PI-88. Many PI-88 variants produced syncytia in cultured cells and contained alterations in gB, including the syncytium-inducing L792P amino acid substitution. Although this phenotype can enhance the lateral spread of HSV in cells, it conferred no virus resistance to PI-88. Some PI-88 variants also contained occasional alterations in gC, gD, gE, gK, and UL24. In conclusion, we found that glycoprotein gG, a mucin-like component of the HSV-2 envelope, was targeted by sulfated oligo- and polysaccharides. This is a novel finding that suggests the involvement of HSV-2 gG in interactions with sulfated polysaccharides, including cell surface glycosaminoglycans.