Varicella-zoster virus (VZV) encodes 69 unique open reading frames, 5 of which do not have herpes simplex virus type 1 (HSV-1) homologs. One of the 5, ORF1, is predicted to encode a protein of 108 amino acids. We identified a 470-base RNA corresponding to ORF1. To determine whether ORF1 encodes a protein, an 11-amino-acid epitope was inserted in frame after the ninth codon of the ORF1 open reading frame. A recombinant virus carrying this epitope expressed a protein that was immunoprecipitated with monoclonal antibody to the epitope. The ORF1 protein was detected in the membrane of infected cells. The size of ORF1 protein expressed in VZV-infected cells was slightly larger than the size expressed by translation in vitro, suggesting that the protein may undergo post-translational modification in infected cells. Insertion of stop codons immediately before the epitope in the ORF1 gene resulted in a recombinant virus that did not express ORF1 protein and that was not growth impaired in cell culture. Thus, ORF1 encodes a protein that localizes to the membrane of VZV-infected cells and that is dispensable for virus growth in vitro.