To minimize the contribution of residual activity associated with the temperature-sensitive (ts) form of ICP8 specified by available ts mutants, deletion mutations in this gene were constructed. Cells permissive for the generation and propagation of ICP8 deletion mutants were first obtained. Vero cells were cotransfected with pKEF-P4, which contains the gene for ICP8, and pSV2neo or a hybrid plasmid containing the G418 resistance gene linked to pKEF-P4. Of the 48 G418-resistant cell lines, 21 complemented ICP8 ts mutants in plaque assays at the nonpermissive temperature. Four of these were examined by Southern blot analysis and shown to contain 1 to 3 copies of the ICP8 gene per haploid genome equivalent. Cell line U-47 was used as the permissive host for construction of ICP8 deletion mutants. In addition to cell lines which complemented ts mutants, two lines, U-27 and U-35, significantly inhibited plaque formation by wild-type virus, contained 30 and 100 copies of the ICP8 gene per haploid genome equivalent, respectively, and expressed large amounts of ICP8 after infection with wild-type virus. At low but not high multiplicities of infection, this inhibition was accompanied by underproduction of viral polypeptides of the early, delayed-early, and late kinetic classes. For construction of deletion mutants, a 780-base-pair XhoI fragment was deleted from pSG18-SalIA, a plasmid which contains the gene for ICP8, to yield pDX. U-47 cells were then cotransfected with pDX and infectious wild-type DNA. Mutant d61, isolated from the progeny of cotransfection, was found to contain both the engineered deletion in the ICP8 gene and an oriL-associated deletion of approximately 55 base pairs. Because d61 contained two mutations, a second mutant, d21, which carried the engineered ICP8 deletion but an intact oriL, was constructed by cotransfection of U-47 cells with wild-type DNA and an SalI-KpnI fragment purified from pDX. Phenotypic analysis of d21 and d61 revealed that they were similar in all properties examined: both exhibited efficient growth in U-47 cells but not in Vero cells; both induced the synthesis of an ICP8 polypeptide which was smaller than the wild-type form of the protein and which, unlike the wild-type protein, was found in the cytoplasm and not the nucleus of infected Vero cells; and nonpermissive Vero cells infected with either mutant failed to express late viral polypeptides.