Experiments were conducted to identify and characterize the equine herpesvirus type 1 (EHV-1) UL11 homologous protein. At early-late times after EHV-1 infection of Rk13 cells several proteins at an M(r) of 8000 to 12,000 were detected using a UL11 protein-specific antiserum. Particularly, an M(r) of 11,000 protein was found abundantly in purified virions and could be assigned to the tegument fraction. As demonstrated by confocal laser scanning microscopy, UL11 reactivity localized predominantly to the trans-Golgi network of infected cells, but was also noted at the plasma membrane, specifically of transfected cells. Deletion of UL11 sequences in EHV-1 vaccine strain RacH (Hdelta11) and in the virulent isolate RacL22 (Ldelta11) resulted in viruses that were able to replicate on noncomplementing cells. It was shown in one-step growth kinetics on Rk13 cells that the reduction of intracellular and of extracellular virus titers caused by the absence of UL11 expression in either virus was somewhat variable, but approximately 10- to 20-fold. In contrast, a marked influence on the plaque phenotype was noted, as mean maximal diameters of plaques were reduced to 23.2% (RacL22) or 34.7% (RacH) of parental virus plaques and as an effect on the ability of RacH to cause syncytia upon infection was noted. It was therefore concluded that the EHV-1 UL11 product is not essential for virus replication in Rk13 cells but is involved in cell-to-cell spread.