The tegument of herpes simplex virus type 1 (HSV-1) virus particles is a complex assemblage of virus proteins whose relative proportions within virions are essentially constant for a particular strain of virus. To examine the processes controlling incorporation into the tegument, we constructed a HSV-1 recombinant that expresses two copies of gene UL49, which encodes the major tegument protein VP22. One copy specifies the unmodified form of VP22 under the control of the native promoter while the second expresses an epitope-tagged version of the protein via the human cytomegalovirus immediate early promoter. In cells infected with the recombinant virus, the overall levels of VP22 synthesized were about fivefold higher than those for wild-type virus, due to the high levels of expression of tagged protein. Analysis of virus particles revealed that the amount of VP22 in the tegument was approximately two- to threefold higher in recombinant virions and L-particles than in particles produced by wild-type virus. These results provide the first evidence that, for certain proteins, the level of polypeptide synthesis can act as a controlling factor for the amount of protein incorporated into tegument.