The herpes simplex virus-1 (HSV-1)-infected cell protein 0 (ICP0) is an E3 ubiquitin ligase implicated in cell cycle arrest and DNA repair inhibition. Convection-enhanced delivery (CED) of either the replication-defective, ICP0-producing HSV-1 mutant, d106, or the recombinant d109, devoid of all viral genome expression, was performed to determine the in vivo efficacy of ICP0 in combination with ionizing radiation (IR) or systemic temozolomide (TMZ) in the treatment of glioblastoma multiforme (GBM). Intracranial U87-MG xenografts were established in athymic nude mice. Animal survival was determined after mice underwent intracranial CED of either the replication-defective d106 or d109 viruses, or Hanks' balanced salt solution (HBSS), before a single session of whole-brain irradiation or TMZ treatment. Median survival for animals that underwent treatment with HBSS alone, d109 alone, d106 alone, HBSS + IR, HBSS + TMZ, d109 + IR, d106 + IR, and d106 + TMZ was 28, 35, 41, 39, 44, 39, 68 (P < 0.01), and 66 days (P < 0.01), respectively. Intracerebral d106 CED resulted in a significant increase in athymic nude mouse survival when combined with IR or TMZ. d106 CED allows for distribution of HSV-1 in human GBM xenografts and persistent viral infection.