The DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT, AGT) is a determinant of the resistance of tumor cells to alkylating anticancer agents that target the O(6) position of guanine. MGMT promoter methylation in tumors is regarded as the most common predictor of the responsiveness of glioblastoma to alkylating agents. However, MGMT promoter methylation status has been investigated mainly by methylation-specific PCR, which is a qualitative and subjective assay. In addition, the actual enzymatic activities associated with the methylation status of MGMT have not been explored. In the present study, MGMT promoter methylation in glioblastomas was quantified by bisulfite pyrosequencing, and its correlation with enzymatic activity was determined using a novel quantitative assay for studying the functional activity of MGMT. MGMT enzymatic activity was assessed using fluorometrically labeled oligonucleotide substrates containing MGMT-specific DNA lesions and capillary electrophoresis to detect and quantify these lesions. In comparison with existing traditional assays, this assay was equally sensitive but less time consuming and easier to perform. MGMT promoter methylation was assessed in 41 glioblastomas by bisulfite pyrosequencing, and five samples with different values were chosen for comparison with enzymatic assays. Bisulfite pyrosequencing using primers designed to work in the upstream promoter regions of MGMT demonstrated high quantitative capability and reproducibility in triplicate measurements. In comparative studies, MGMT promoter methylation values obtained by bisulfite pyrosequencing were inversely proportional to the measured enzymatic activity. The present results indicate that the quantification of MGMT methylation by bisulfite pyrosequencing represents its enzymatic activity and thus, its therapeutic responsiveness to alkylating agents.