Previous studies have shown that cell-associated herpes simplex virus (HSV) glycoprotein gD can interfere with infection of the cells by HSV and other alphaherpesviruses and that HSV mutants resistant to this gD-mediated interference can be isolated. Here we report that HSV mutants selected for resistance to gD-mediated interference are altered in specific infectivity for cells that do not express gD. Two independently derived mutants were shown to be impaired in ability to infect HEp-2 cells and enhanced in ability to infect Chinese hamster ovary cells, compared with the wild-type parental strain. The mutants were not significantly different from the parental strain in ability to bind to cells but differed in a postbinding step required for infectivity, probably penetration. The two mutants were shown to have different amino acid substitutions (Q27P and Q27R) in gD. Marker transfer experiments demonstrated that the resistance to gD-mediated interference as well as the altered infectivities resulted from these amino acid substitutions. Thus, small changes in gD structure can not only confer resistance to gD-mediated interference but also alter the relative efficiencies with which HSV penetrates into different cell types.