The production of DNA interstrand crosslinks (ISC) by BCNU and other bifunctional alkylators and the effects of these drugs on the repair of radiation-induced DNA-single strand breaks (SSB) were studied in two human glioblastoma used to assess both DNA-ISCs and DNA-SSBs. BCNU-treated UWR2 and UWR3 cells showed a significant BCNU dose-dependent increase in radiation-induced DNA-SSBs at 6 hrs post-drug treatment, and at 100 microM BCNU DNA-ISC was completely masked in UWR2 cells. There was no enhancement of radiation-induced DNA-SSBs in both cell lines after treatment with cis-DDP, CHZ, or MNU. In the capillary clonogenic cell assay, UWR2 cells were 3.2 times more resistant than UWR3 cells; 0(6)-methylguanine-DNA methyltransferase activity was also 1.8 times higher in UWR2 than in UWR3. Our data suggest caution in the use of the standard alkaline elution technique (with 6 hrs between drug exposure and irradiation) to measure BCNU-induced DNA-ISC induction in highly BCNU-resistant cell lines. We provide evidence that the synergism between BCNU and radiation in the generation of DNA-SSBs is the result of low DNA-SSB repair capacity of the cells, and is further potentiated by the carbamoylating action of BCNU.