The mouse L-cell mutant gro29 is defective for egress of herpes simplex virus type 1 (HSV-1) virions and is significantly reduced in HSV-1 glycoprotein export (B. W. Banfield and F. Tufaro, J. Virol. 64:5716-5729, 1990). In this report, we demonstrate that pseudorabies virus (PRV), a distantly related alphaherpesvirus, shows a distinctive set of defects after infection of gro29 cells. Specifically, we identify defects in the rate and extent of viral glycoprotein export, infectious particle formation, plaque formation, and virus egress. The initial rate of viral glycoprotein synthesis was unaffected in gro29 cells, but the extent of export from the endoplasmic reticulum to the Golgi apparatus was impaired and export through the Golgi apparatus became essentially blocked late in infection. Moreover, by using a secreted variant of a viral membrane protein, we found that export from the Golgi apparatus out of the cell was also defective in gro29 cells. PRV does not form plaques on gro29 monolayers. A low level of infectious virus is formed and released early after infection, but further virus egress is blocked. Taken together, these observations suggest that the gro29 phenotype involves either multiple proteins or a single protein used at multiple steps in viral glycoprotein export and virus egress from cells. Moreover, this host cell protein is required by both HSV and PRV for efficient propagation in infected cells.