Previous studies have suggested that the U(L)17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZ expression cassette in place of 1,490 bp of the 2,109-bp U(L)17 open reading frame [HSV-1(deltaU(L)17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5' end of the U(L)17 open reading frame [HSV-1(U(L)17-stop)] were plaque purified on engineered cell lines containing the U(L)17 gene. A virus derived from HSV-1(U(L)17-stop) but containing a restored U(L)17 gene was also constructed and was designated HSV-1(U(L)17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(deltaU(L)17) nor HSV-1(U(L)17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(deltaU(L)17) compared to wild-type virus show no detectable differences. These data indicate that the U(L)17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the U(L)17 gene product, an anti-U(L)17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent Mr 77,000 and weakly with a protein of apparent Mr 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted U(L)17 protein. We therefore conclude that the U(L)17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.