Previous studies have identified requirements for the binding of herpes simplex virus type 1 (HSV-1) to cells, including the presence of particular glycoproteins in the virion envelope (gC or gB) and the presence of particular glycosaminoglycan chains (principally heparan sulfate) on cell surface proteoglycans. We show here that neomycin, a known inhibitor of HSV infection, blocked early events in HSV infection by two mechanisms: partial inhibition of the gC-dependent binding of virions, but not the gB-dependent binding, and inhibition of events that occurred after the binding of virus to cells. Near-maximal (but incomplete) inhibition of virus binding occurred at low concentrations of neomycin (1 mM) for wild-type and gB-negative virions only. Neomycin also inhibited the binding of isolated gC to cells at a similar concentration. Concentrations of neomycin as high as 50 mM had little or no effect on the binding of gC-negative virions to cells. Nevertheless, neomycin significantly inhibited infection by both wild-type and gC-negative virions, at concentrations greater than 10 mM, indicating that the inhibition at higher doses was not due to effects on virus binding. The effects of neomycin on virus binding suggest that gC (but not gB) and neomycin compete for binding to similar structural features of cell surface heparan sulfate.