Pancreatic adenocarcinomas rarely respond to radiation or chemotherapy, indicating that a large percentage of these tumors possess complex mechanisms of resistance. The failure of alkylating agents, such as carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea; BCNU], lomustine [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; CCNU], and streptozotocin, to yield consistent therapeutic results further suggests that one of these mechanisms may be the high expression of O6-methylguanine-DNA methyltransferase (MGMT). All 12 human pancreatic ductal adenocarcinomas assayed for MGMT activity showed unusually high levels, implying that these malignancies are efficient in repairing genotoxic O6-alkylguanine lesions induced by methylating (streptozotocin) and 2-chloroethylating (BCNU and CCNU) chemotherapeutic genotoxic agents. Immunohistochemical analysis of an additional 15 pancreatic tumors showed that high levels of MGMT protein reside in the nucleus and the cytoplasm of malignant cells. Both nuclear and cytoplasmic staining were absent in hyperplastic duct epithelium, but staining was invariably present in moderate to highly dysplastic foci and especially strong in invasive components of the tumor. With the exception of lymphocytes that were MGMT positive, acinar, ductal, and islet cells did not stain for MGMT in histologically normal pancreata. These data indicate that MGMT activity is up-regulated in dysplastic epithelium, and its expression increases during tumor progression, reaching the highest levels in the invasive components of the tumor. Resistance of pancreatic tumor cells to alkylating agents was verified with four pancreatic tumor cell lines. CAPAN-2, CFPAC-1, PANC-1, and MIAPaCa-2, having MGMT levels of 1800, 987, 700, and 880 fmol/mg protein, respectively, were resistant to BCNU, but their resistance declined sharply following pretreatment with the MGMT inhibitor O6-benzylguanine (O6-BG). On the other hand, PANC-1 and MI-APaCa-2 could not be eradicated with N-methylnitrosourea (MNU) at concentrations as high as 2 mm, even when pretreated with O6-BG. These two lines were shown to be modified genetically in microsatellite sequences by MNU and are believed to have a defective mismatch repair system, which may explain their resistance to methylating agents. Failure of pancreatic tumors to respond to nitrosoureas is related to high levels of MGMT expression and in some cases to genomic instability. However, these tumors can be sensitized to chloroethylating drugs and eradicated following the elimination of MGMT activity by O6-BG or homologous MGMT inhibitors.