Glycoproteins gE and gG of bovine herpesvirus 1 (BHV-1) are involved in viral cell-to-cell transmission. We have compared the subcellular localizations of gE and gG and examined the cell-to-cell adherence of bovine kidney (MDBK) cells infected with BHV-1 mutants lacking gE or gG. In BHV-1-infected MDBK cells, gE was observed at cell junctions but did not localize at apical or basal plasma membranes. BHV-1 gG was primarily found in the cytoplasm and was also observed at boundaries among infected cells. During the infection with wild-type or gE-negative BHV-1, the filamentous actin and the adherent junctional proteins accumulated at the cell junctions. In contrast, cell junctions of MDBK cells infected with gG-negative BHV-1 were loosened, and the junctional proteins and BHV-1 gE were distributed in the cytoplasm. These data indicate that BHV-1 gG facilitates viral cell-to-cell spread by maintaining the cell-to-cell junctions among the infected cells.