We studied the expression of O(6)-methylguanine-DNA methyltransferase (O(6)-MGMT), P-glycoprotein (Pgp), and multidrug resistance protein-1 (MRP-1) in 23 glioblastomas using RT-PCR, methylation-specific PCR, and immunohistochemistry, and analyzed their association with overall patient survival. Univariate analysis of collected data demonstrated that the expressions of O(6)-MGMT and MRP-1 detected by immunohistochemistry, in addition to the consistent factors, including preoperative Karnofsky performance scale (KPS), radical surgery, and tumor location and extension, were significant prognostic factors for the overall survival (OS) of patients with glioblastoma, who received nimustine (ACNU)-based chemotherapy in association with surgery and radiotherapy. Among them, following multivariate analysis, preoperative KPS, radical surgery, tumor location, and the expression of O(6)-MGMT remained as significant prognostic factors. These findings suggest that immunohistochemical analysis of O(6)-MGMT in patients with glioblastoma can be a useful method to predict the effects of chemotherapy and identify alternative chemotherapeutic regimens for O(6)-MGMT-positive patients.