Purpose: Glioblastoma multiforme is a deadly primary brain cancer. Because the tumor kills due to recurrences, we tested the hypothesis that a new treatment would lead to immunological memory in a rat model of recurrent glioblastoma multiforme. Experimental Design: Wedeveloped a combined treatment using an adenovirus (Ad) expressing fms-like tyrosine kinase-3 ligand (Flt3L),which induces the infiltration of immune cells into the tumor microenvironment, and an Ad expressing herpes simplex virus-1-thymidine kinase (TK), which kills proliferating tumor cells in the presence of ganciclovir. Results: This treatment induced immunologicalmemory that led to rejection of a second glioblastoma multiforme implanted in the contralateral hemisphere and of an extracranial glioblastomamultiforme implanted intradermally. Rechallenged long-termsurvivors exhibited anti-glioblastoma multiforme-specific T cells and displayed specific delayedtype hypersensitivity. Using depleting antibodies,we showed that rejection of the second tumor was dependent on CD8+ T cells. Circulating anti©\glioma antibodies were observed when glioblastoma multiforme cells were implanted intradermally in naïve rats or in long-termsurvivors. However, rats bearing intracranial glioblastomamultiforme only exhibited circulating antitumoral antibodies upon treatment with Ad-Flt3L + Ad-TK. This combined treatment induced tumor regression and release of the chromatin-binding protein high mobility group box 1 in two further intracranial glioblastoma multiforme models, that is, Fisher rats bearing intracranial 9L and F98 glioblastoma multiforme cells. Conclusions: Treatment with Ad-Flt3L + Ad-TK triggered systemic anti-glioblastoma multiforme cellular and humoral immune responses, and anti-glioblastoma multiforme immunological memory. Release of the chromatin-binding protein high mobility group box 1 could be used as a noninvasive biomarker of therapeutic efficacy for glioblastoma multiforme. The robust treatment efficacy lends further support to its implementation in a phase I clinical trial. © 2009 American Association for Cancer Research.