Abstract Epigenetic silencing of the O6-methylaguanine DNA methyl transferase (MGMT) gene by promoter hypermethylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. Quantitative methylation specific PCR (Q-MSP) suggests that not all cells in a glioblastoma exhibiting MGMT methylation carry a methylated MGMT allele, even after adjustment for tumor cell content. This raises the question of the clinically relevant methylation threshold for predicting response to therapy for treatment stratification. There are several potential reasons: heterogeneity of glioblastoma; contaminating normal tissue; a heterogenous methylation pattern not correctly detectable by the assay, or presence of MGMT methylation in distinct subpopulations of cells, namely tumor initiating cells. In order to address the question we investigated the degree and pattern of MGMT promoter methylation in 10 paired samples of glioblastoma and spheres derived thereof using Q-MSP and methylation specific clone sequencing. We observed that in MGMT methylated glioblastoma, the percentage of methylated alleles ranged from 10 to 90%. In contrast, in most of the respective glioblastoma derived spheres, cultured under stem cell conditions, methylated alleles were highly enriched (10/10 clones), even if both MGMT-alleles were retained. The methylation pattern was conserved in the spheres, even when passing them in nude mice. Most importantly, this hypermethylation was associated with complete loss of MGMT expression. For one case we observed only 50% methylated clones in the sphere fraction, associated with low expression of MGMT. Taken together, our data suggest that in MGMT methylated glioblastoma, cells with MGMT promoter hypermethylation are enriched in the sphere fraction which supposedly comprises cancer initiating cells. Thus, even a low percentage of MGMT methylation in a glioblastoma could be predictive marker for a benefit from alkylating agent therapy. Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4927.