Recent evidence from our laboratory and from others suggested that pretreatment with alpha-difluoromethylornithine (DFMO) sensitizes some human and rodent tumor cell lines to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Many human tumor cells are resistant to chloroethylnitrosourea-induced DNA interstrand cross-linking and cell kill due to their high levels of the DNA repair protein O6-alkylguanine DNA alkyltransferase. We therefore investigated DFMO-mediated sensitization to BCNU in BCNU-sensitive and -resistant cells. Colony formation assays were used to compare BCNU cytotoxicity in DFMO-pretreated and control cultures of two colon tumor lines, HT-29 cells, which have high alkyltransferase levels and thus are BCNU-resistant, and BE cells, which are deficient in this repair capacity and thus are BCNU-sensitive. Polyamine depletion significantly enhanced BCNU cytotoxicity only for the repair-proficient HT-29 cell line. BE cells were 40-fold more sensitive to BCNU than were HT-29 cultures. However, in BE cells, no effect of polyamine depletion was found on cellular response to BCNU treatment at 72 h after DFMO treatment. Reverse-phase high-performance liquid chromatography assays of polyamine concentrations in cell extracts verified that DFMO produced comparable degrees of polyamine depletion for both cell lines. DNA alkaline elution analysis was used to monitor BCNU-induced formation of DNA single strand breaks, DNA interstrand cross-links, and DNA-protein cross-links. Equal concentrations of BCNU produced similar levels of strand breaks and DNA-protein cross-links in DFMO-pretreated and control cultures for both cell lines. These data suggest that DNA in polyamine-deficient HT-29 and BE cells is not more accessible to BCNU than is DNA in controls. No DNA interstrand cross-links were detected in either DFMO-pretreated or control HT-29 cells after BCNU treatment. Further, in BE cells which accumulate BCNU-induced DNA interstrand cross-links, no increase in the measureable levels of cross-links resulted from polyamine deficiency. Our observations suggest that mechanisms other than increased DNA interstrand cross-link formation may be mediating the enhanced efficacy of BCNU in polyamine-deficient HT-29 cell cultures. Our findings may also imply that cellular targets for BCNU other than DNA damage may be responsible for DFMO-induced chemosensitization in the repair-proficient cells.