Background: Cationic liposome-mediated interferon-beta (IFN-b) gene transfer has been found to induce regression of experimental glioma. We performed a pilot clinical trial to evaluate safety and effectiveness of this IFN-b gene therapy in five patients with malignant glioma (glioblastoma multiforme or anaplastic astrocytoma). Two patients showed more than 50% reduction and others had stable disease 10 weeks after the treatment initiation. Autopsy: Autopsy revealed that tumor tissues showed dramatic changes after therapy in all patients. Many tumor cells showed necrotic changes, and immunohistochemistry identified many CD8-positive lymphocytes and macrophages infiltrating in tumor and surrounding tissues, probably resulting from therapeutic effect. Simultaneously, the number of MIB-1-positive cells was notably decreased. No adverse findings associated with the clinical trial were pathologically observed. Microarray: In order to identify alterations in gene expression in brain tumors 2 weeks after gene therapy trial, we used microarray technology, which allows us to examine the expression of a large number of genes simultaneously. Interestingly, using hierarchical clustering and principal component analysis, 5 series of gene therapy trial were classified according to response to IFN gene therapy. There were changes in gene expression which could be predicted on the basis of previous studies. It confirms the validity of the methods. Moreover, novel patterns of altered gene expression were identified, suggesting the involvement of pathways not previously described as involved. Conclusions: This study suggests the feasibility and safety of IFN-b gene therapy, which may become an important treatment options for patients with malignant glioma.