Mutants of herpes simplex virus type 1 (HSV-1) strain SC16, lacking each of the dispensable glycoproteins C, G, E, I or J, were examined for their ability to infect the apical or basolateral surfaces of polarized human epithelial cells. None of the mutants was significantly different from the wild-type parent when assayed on either surface. Since a previous report had demonstrated that glycoprotein C (gC) was necessary for the infection of apical surfaces of polarized epithelium, a second gC-negative mutant was constructed on a background of HSV-1 strain HFEM. This mutant displayed no phenotype when assayed on the apical surface. Furthermore, neither gC-negative mutant differed from its wild-type parent in its adsorption kinetics or specific infectivity on non-polarized Vero cells, a result which is inconsistent with the view that interactions between gC and cell surface proteoglycans constitute the initial adsorption process. Our findings thus conflict with previous reports and suggest that proposed functions of HSV-1 gC in the infection of polarized and non-polarized cells may be strain-dependent.