Earlier studies have shown that genetically engineered herpes simplex viruses (e.g., HSV-1) are effective in killing malignant tumor cells both in vitro and in various murine tumor models. This report focuses on a panel of five genetically engineered vital mutants of the γ134.5 gene, which was shown previously to cause reduction in viral replication and associated neurovirulence of HSV. These include R3616, which has both copies of γ134.5 deleted, R4009, which has a stop codon inserted after codon 28 in both copies of the γ134.5 gene, R849, which contains a lacZ gene inserted in place of the γ134.5, R908, which lacks 41 codons in frame after codon 72 of the γ134.5, and R939, which carries a stop codon precluding the translation of the COOH-terminal domain of the γ134.5 gene. We report the following: (a) all five mutant HSVs were avirulent in experimental animals but were cytotoxic for human tumor cells in vitro and in vivo; (b) the γ134.5- HSV replicated in human glioma cells almost as efficiently as wild-type HSV-1(F) based on replication assays, in situ hybridization for viral DNA, and expression of infected cell protein 27; (c) capacity of mutant HSVs to kill human cells derived from glioblastoma multiforme (CH-235MG, D-37MG, D-54MG, D-65MG, U-251MG, U-373MG, and NK-MG-1), anaplastic astrocytoma (Hs-683), anaplastic glioma (U-87MG and U-138MG), gliosarcoma (D-32GS), or normal human astrocytes demonstrated that glioma cells varied in their susceptibility to HSV-mediated cytotoxicity and that cultured astrocytes were two to three orders of magnitude less susceptible to killing than were malignant glia; and (d) scid mice, which received 0.5 or 5 x 106 plaque-forming units of R4009, either were coinoculated at the time of intracranial transplantation with 106 U251MG or D-54MG human glioma cells or received the cells intratumorally 5 days after tumor induction and experienced significant increases in median survivals, with no histopathological indication of an infectious encephalitic process. Genetically engineered γ134.5- HSV mutants appear to be a potentially safe biotherapeutic agent for experimental treatment of uniformly fatal malignant brain tumors.