The role of the nonessential glycoproteins gI, gp63, and gIII in the release of pseudorabies virus from different cell lines was investigated. We show that these glycoproteins may have a beneficial or deleterious effect on virus release depending on the type of cell in which the virus is grown. Inactivation of the genes encoding either gI, gp63, or gIII has no detectable effect on virus release from rabbit kidney cells. Inactivation of gI or gp63 strongly promotes virus release from chicken embryo fibroblasts, whereas inactivation of gIII reduces virus release from these cells. A defect in both gI and gIII or in both gp63 and gIII diminishes virus release from rabbit kidney cells but improves release from chicken embryo fibroblasts. We demonstrate that all three nonessential glycoproteins contribute to one specific aspect of viral growth, namely, virus release, and that they affect virus release in conjunction with each other. Furthermore, our results show that the manifestation of the role of each of these viral functions in virus growth may differ in different cell types, i.e., that release is affected by these viral functions in conjunction with some unknown cellular function.