Although the expression O6-methylguanine-DNA methyltransferase (MGMT) is an important hallmark for decision of nitrosourea chemotherapy for glioma patients, no immunohistochemical method for analysis of MGMT has been standardized yet. Gliomas usually contain non-neoplastic cells even deep in the tumor. It is not known which of these components expresses MGMT. To clarify this point, we investigated MGMT expression in the non-neoplastic cells in autopsy and surgical specimens by immunohistochemistry. High grade gliomas were also studied to find a cut-off point for treatment decision. MGMT immunohistochemistry in the normal brain or brain with non-neoplastic disease revealed nuclear staining in some endothelial cells, inflammatory cells, ependymal cells, astrocytes and oligodendroglias. Some cells were double stained with CD68 (macrophages or microglias). The neurons were consistently MGMT-negative. High grade gliomas always contained an MGMT-positive non-neoplastic component. Although, the endothelial cells were easily distinguished from the neoplastic cells, other cells were often mistaken for tumor cells. The population of MGMT-positive non-neoplastic cells was usually less than 10%. We set a cut off-point at 10% between the positive and negative groups because the statistical difference in the overall survival was most distinct at this value. In 51 high grade glioma patients, who received both radiotherapy and chemotherapy with nimustine (ACNU), the median overall survival of the MGMT-negative group (23 months) was significantly longer than that of the MGMT-positive group (14 months) (P < 0.009). Multivariate analysis revealed that the negative MGMT expression was a significant prognostic variable next to the degree of surgical removal for the overall survival. In the MGMT-positive group, addition of platinum-based chemotherapy did not improve the survival.