O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that interferes with chemotherapeutic effect of alkylating agents. We performed in situ detection of MGMT mRNA utilizing the nested RT-PCR method in tissue sections (nested in situ RT-PCR). We analyzed 34 samples of paraffin-embedded astrocytic tumor tissue sections with this method [3 astrocytomas, 14 anaplastic astrocytomas (AA), and 17 glioblastoma multiformes (GBM)]. Twenty-five cases (73.5% of all cases) were positive for MGMT either with our method or immunohistochemistry (IHC). Moreover, with our method >25% of the cells in the tumor tissue expressed MGMT in contrast to >4% with IHC among the population of MGMT positive cases. Our method was significantly more sensitive than IHC (p=0.0004). The present results suggest that potentially there is a greater population of MGMT positive cells in astrocytic tumor tissues than the one evaluated with IHC. These findings suggest that the >25% of the MGMT positive cells are involved in the interference with the chemotherapeutic effect of alkylating agents. The MGMT expressing cell population was markedly decreased in GBM compared with AA (26.1% vs 62.1%). The main reason for this marked decrease was that MGMT was expressed in only 9 of 17 cases of GBM in contrast to all AA cases that expressed MGMT. This result suggests that there are potentially two populations of GBM on the basis of MGMT expression, in which the negative population might be mainly composed of de novo GBM. Therefore, it is suggested that our method is practically useful to detect any drug resistance gene product with high sensitivity and would provide a chance to evaluate the chemotherapeutic effect of any agents in an individual patient based manner.