HSV infection is initiated by binding of viral envelope gC to cell surface heparan sulfate (HS). HS and related glycoaminoglycan heparin are a family of complex carbohydrates composed of repeating disaccharide units modified by sulfation. To define specific disaccharide composition of HS important for gC binding, purified gC was generated. The gC bound to heparin-Sepharose and was eluted at a concentration of 0.2M-0.5M NaCl. Also, the gC bound epithelial cells (Vero and HEp-2) in a heparin-inhibitable fashion. To evaluate the number of saccharide units important for gC's binding, competition studies were performed. 35S labeled gC was incubated with heparin-Sepharose beads in the presence or absence of either oligosaccharides of varying chain length, low molecular weight heparin (LMWH), or heparin. Oligosaccharides of up to 10 saccharide units failed to compete with heparin-Sepharose for binding to gC, however, LMWH competed similar to heparin. These results indicate that gC interacts with an oligosaccharide of greater than 10 and fewer than 20 saccharide unit. To isolate gC-specific polysaccharides, gC was immobilized on a concanavalin A-Sepharose (conA) column. Heparin or HS was passed over the gC-conA column, and polysaccharides that bound to gC were eluted with a salt gradient, and identified using a modified carbazole-borate reaction. Two populations of polysaccharides were eluted from the column at approximately 175 and 300 mM, respectively. To test if these fractions had biologic activity, binding studies were performed. The 300mM fraction inhibited binding of HSV-1 to Vero cells similar to heparin. In the presence of 2ug/ml of carbohydrate, 34±3.5% or 32±1.1% of virus remained bound to cells in the presence of heparin or the 300mM fraction, respectively. A log more carbohydrate was required to achieve the same level of inhibition using the 175mM fraction. In conclusion, biologically active gC of HSV-1 interacts with polysaccharide chains of more than 10 saccharide units with 2 distinct affinities. Further characterization of the 300mM and 175mM polysacchride fractions may aid in identifying a competitive inhibitor of viral attachment with potential as a topical therapy.