UNLABELLED: The alphaherpesvirus pseudorabies virus (PRV) encodes a single immediate early gene called IE180. The IE180 protein is a potent transcriptional activator of viral genes involved in DNA replication and RNA transcription. A PRV mutant with both copies of IE180 deleted was constructed 20 years ago (S. Yamada and M. Shimizu, Virology 199:366-375, 1994, doi:10.1006/viro.1994.1134), but propagation of the mutant depended on complementing cell lines that expressed the toxic IE180 protein constitutively. Recently, Oyibo et al. constructed a novel set of PRV IE180 mutants and a stable cell line with inducible IE180 expression (H. Oyibo, P. Znamenskiy, H. V. Oviedo, L. W. Enquist, A. Zador, Front. Neuroanat. 8:86, 2014, doi:10.3389/fnana.2014.00086), which we characterized further here. These mutants failed to replicate new viral genomes, synthesize immediate early, early, or late viral proteins, and assemble infectious virions. The PRV IE180-null mutant did not form plaques in epithelial cell monolayers and could not spread from primary infected neurons to second-order neurons in culture. PRV IE180-null mutants lacked the property of superinfection exclusion. When PRV IE180-null mutants infected cells first, subsequent superinfecting viruses were not blocked in cell entry and formed replication compartments in epithelial cells, fibroblasts, and neurons. Cells infected with PRV IE180-null mutants survived as long as uninfected cells in culture while expressing a fluorescent reporter gene. Transcomplementation with IE180 in epithelial cells restored all mutant phenotypes to wild type. The conditional expression of PRV IE180 protein enables the propagation of replication-incompetent PRV IE180-null mutants and will facilitate construction of long-term single-cell-infecting PRV mutants for precise neural circuit tracing and high-capacity gene delivery vectors. IMPORTANCE: Pseudorabies virus (PRV) is widely used for neural tracing in animal models. The virus replicates and spreads between synaptically connected neurons. Current tracing strains of PRV are cytotoxic and kill infected cells. Infected cells exclude superinfection with a second virus, limiting multiple virus infections in circuit tracing. By removing the only immediate early gene of PRV (called IE180), the mutant virus will not replicate or spread in epithelial cells, fibroblasts, or neurons. The wild-type phenotype can be restored by transcomplementation of infected cells with IE180. The PRV IE180-null mutant can express fluorescent reporters for weeks in cells with no toxicity; infected cells survive as long as uninfected cells. Infection with the mutant virus allows superinfection of the same cell with a second virus that can enter and replicate. The PRV IE180-null mutant will permit conditional long-term tracing in animals and is a high-capacity vector for gene delivery.