Promoter hypermethylation and histone deacetylation are common epigenetic mechanisms implicated in the transcriptional silencing of tumor suppressor genes in human cancer. We treated two immortalized glioma cell lines, T98 and U87, and 10 patient-derived primary glioma cell lines with trichostatin A (TSA), a histone deacetylase inhibitor, or 5-aza-2'-deoxycytidine (5-AzaC), a DNA methyltransferase inhibitor, to comprehensively identify the cohort of genes reactivated through the pharmacologic reversal of these distinct but related epigenetic processes. Whole-genome microarray analysis identified genes induced by TSA (653) or 5-AzaC treatment (170). We selected a subset of reactivated genes that were markedly induced (greater than two-fold) after treatment with either TSA or 5-AzaC in a majority of glioma cell lines but not in cultured normal astrocytes. We then characterized the degree of promoter methylation and transcriptional silencing of selected genes in histologically confirmed human tumor and nontumor brain specimens. We identified two novel brain expressed genes, BEX1 and BEX2, which were silenced in all tumor specimens and exhibited extensive promoter hypermethylation. Viral-mediated reexpression of either BEX1 or BEX2 led to increased sensitivity to chemotherapy-induced apoptosis and potent tumor suppressor effects in vitro and in a xenograft mouse model. Using an integrated approach, we have established a novel platform for the genome-wide screening of epigenetically silenced genes in malignant glioma. This experimental paradigm provides a powerful new method for the identification of epigenetically silenced genes with potential function as tumor suppressors, biomarkers for disease diagnosis and detection, and therapeutically reversible modulators of critical regulatory pathways important in glioma pathogenesis.