Initial contact between herpesviruses and host cells is mediated by virion envelope glycoproteins which bind to cellular receptors, In several alphaherpesviruses, the nonessential glycoprotein gC has been found to interact with cell surface proteoglycans, whereas the essential glycoprotein gD is involved in stable secondary attachment, In addition, gD is necessary for penetration, which involves fusion between virion envelope and cellular cytoplasmic membrane, As opposed to other alphaherpesvirus gD homologs, pseudorabies virus (PrV) gD is not required for direct viral cell-to-cell spread, Therefore, gD(-) PrV can be passaged in noncomplementing cells by cocultivating infected and noninfected cells, Whereas infectivity was found to be strictly cell associated in early passages, repeated passaging resulted in the appearance of infectivity in the supernatant, finally reaching titers as high as 10(7) PFU/ml (PrV gD(-) Pass), Filtration experiments indicated that this infectivity was not due to the presence of infected cells, and the absence of gD was verified by Southern and Western blotting and by virus neutralization. Infection of bovine kidney cells constitutively expressing PrV gD interfered with the infectivity of wild type PrV but did not inhibit that of PrV gD(-) Pass, Similar results were obtained after passaging of a second PrV mutant, PrV-376, which in addition to gD also lacks gG, gI, and gE, Penetration assays demonstrated that PrV gD(-) Pass entered cells much more slowly than wild-type PrV. In summary, our data demonstrate the existence of a gD-independent mode of initiation of infection in PrV and indicate that the essential function(s) that gD performs in wild-type PrV infection can be compensated for after passaging. Therefore, regarding the requirement for gD, PrV seems to be intermediate between herpes simplex virus type I, in which gD is necessary for penetration and cell-to-cell spread, and varicella-zoster virus (VZV), which lacks a gD gene, Our data show that the relevance of an essential protein can change under selective pressure and thus demonstrate a way in which VZV could have evolved from a PrV-like ancestor