The cytoplasmic domains of several herpesviral glycoproteins encompass potential intracellular sorting signals. To analyse the function of the cytoplasmic domains of different pseudorabies virus (PrV) glycoproteins, hybrid proteins were constructed consisting of the extracellular and transmembrane domains of envelope glycoprotein D (gD) fused to the cytoplasmic tails of gB, gE or gM (designated gDB, gDE and gDM), all of which contain putative endocytosis motifs. gD is a type I membrane protein required for binding to and entry into target cells. Localization of hybrid proteins compared to full-length gB, gE and gM as well as carboxy-terminally truncated variants of gD was studied by confocal laser scanning microscopy. The function of gD hybrids was assayed by trans-complementation of a gD-negative PrV mutant. The carboxy-terminal domains of gB and gM directed a predominantly intracellular localization of gDB and gDM, while full-length gD and a tail-less gD mutant (gDc) were preferentially expressed on the cell surface. In contrast gDE, and a gDB lacking the putative gB endocytosis signal (gDB Delta 29), were predominantly located in the plasma membrane. Despite the different intracellular localization, all tested proteins were able to complement infectivity of a PrV gD(-) mutant. Cells which stably express full-length gD and plasma-membrane-associated gD hybrids exhibit a significant resistance to PrV infection, while cells expressing predominantly intracellularly located forms do not. This suggests that the assumed sequestration of receptors by gD, which is supposed to be responsible for the interference phenomenon, occurs at the cell surface.