Pseudorabies virus (PrV) infected cells in suspension are able to adhere to a monolayer of uninfected cells by means of PrV glycoproteins expressed at the outer cell membrane, with gB and gC playing a major role as ligands and a heparinlike substance as receptor. In order to investigate the role of gD in this process and subsequent transmission of infectivity to contact cells, experiments with a gD deletion mutant, heparin and a monoclonal antibody (Mab) against gD were performed. The first indication that gD is active during cell adhesion was found by the observation that the binding of gD- PrV infected cells was five times weaker than that of wild type (WT) PrV infected cells. Further evidence was given by the use of a Mab against gD. Preincubation of WT PrV infected cells with this Mab led to a reduction of the percentage adhering cells from 69% to 49%. The same Mab inhibited the heparin independent and heparin resistant binding of WT PrV infected cells indicating that gD is important during both processes. Furthermore, it was demonstrated in a plaque assay that, after contact with a monolayer, gD- PrV infected cells in suspension were able to induce plaques with an efficiency of 1%. In conclusion, we can state that beside the interaction of the ligands gB and gC with a heparinlike receptor also the interaction of gD with a receptor which differs from a heparinlike substance mediates the binding of WT PrV infected cells to uninfected cells and that gD is not essential for the subsequent cell-to-cell spread of the virus.