The aim of this study was to determine if there was an association between expression of toll-like receptor 9 (TLR9) in glioblastoma tissue and patient outcome in glioblastoma multiforme. Further, we characterized the direct in vitro effects of the TLR9 agonist, CpG oligodeoxynucleotide (ODN), commonly used as a vaccine adjuvant in cancer immunotherapy, on glioma cells. TLR9 expression was assessed using immunohistochemical techniques, and enzyme-linked immunosorbent assays were used to investigate the expression of other proteins in glioma cells relevant to immunotherapy. There was a highly significant difference in both progression-free survival and overall survival between TLR9+ and TLR9- patients, with poorer outcome in TLR9+ patients. In in vitro glioma cells, there was a positive correlation between the protein levels of TLR9 and both matrix metalloproteinase (MMP)-2 and MMP-9 (p < 0.05), but no relationship between TLR9 levels and levels of interleukin-6, transforming growth factor-beta 2 or signal transducer and activator of transcription (STAT)-3 (p > 0.05). Our data indicate that expression of TLR9 correlates with shorter progression-free survival and overall survival in patients with glioblastoma multiforme. Our findings also indicate that caution is warranted when directly injecting the TLR9 agonist CpG ODN into glioma tissues as part of glioma immunotherapy. Because the CpG ODN is a TLR9 agonist, we recommend caution when using CpG ODN in immunotherapy. (C) 2011 Elsevier Ltd. All rights reserved.