Herpes simplex viruses encode several glycoproteins dispensable for infection and replication in cell culture. Evidence is presented that there exist at least two pathways for viral attachment to cells, i.e., one mediated by the dispensable glycoprotein C (gC) and one independent of that glycoprotein. Thus, whereas the polycations neomycin and polylysine inhibit attachment but not entry of already attached herpes simplex virus 1 (HSV-1) into baby hamster kidney (BHK) cell line, they have no effect on HSV-2 attachment to the same cells (N. Langeland, H. Holmsen, G.R. Lilehaug, and L. Haarr, 1987, J. Virol. 61, 3388-3393; N. Langeland, L.J. Moore, H. Holmsen, and L. Haarr, 1988, J. Gen. Virol. 69, 1137-1145). We report that (i) analyses of intertypic HSV-1 X HSV-2 recombinants indicated that the HSV-2 locus which confers ability to infect BHK cells in the presence of neomycin or polylysine comaps with the gene specifying gC but not with or near the genes specifying the other viral glycoproteins (gB, gD, gE, and gG, and gI), (ii) the smallest HSV-2 DNA fragment capable of transferring this function to HSV-1 was a 2880-bp Sa/l fragment encoding the entire gC (UL44 open reading frame) gene, 515 bp of coding sequences from the UL43 open reading frame and 393 bp of coding sequences from the UL45 open reading frame, but analyses of the recombinant virus DNA excluded UL43 and most of the UL45 sequences, and (iii) definitive evidence that HSV-2 gC confers upon HSV the capacity to infect BHK cells in the presence neomycin or polylysine emerged from studies showing that site-specific mutagenesis which inactivated the gene yielded a recombinant whose attachment to BHK cells was blocked by the polycations. We conclude that in BHK cells there exists in addition to the pathway blocked by neomycina and polylysine a pathway which is parallel and HSV-2 gC dependent.