Glioma, and in particular high-grade astrocytoma termed glioblastoma multiforme (GBM), is the most common primary tumor of the brain. Epigenetic silencing of the MGMT (O(6)-methylguanine-DNA Methyl transferase) DNA repair gene by promoter methylation compromises DNA repair and has been associated with longer survival in patients with GBM who receive alkylating agents. The methylation status of the MGMT promoter is determined by methylation-specific polymerase chain reaction analysis (MSP). This protocol is often challenging with GBM specimens, because of extensive necrosis and scarcity of malignant cells. The objective of this study was to develop a reliable, clinically validated assay for detection of epigenetic silencing of the MGMT gene using formalin-fixed, paraffin-embedded brain tumor resections and methylation-specific PCR.