Data obtained from clinical samples suggest that non-P-glycoprotein mechanisms of multidrug resistance are likely to be important in small cell lung cancer. The H69AR cell line was derived from the H69 small cell lung cancer cell line by selection in doxorubicin (adriamycin) and does not overexpress P-glycoprotein as detected by monoclonal antibody C219 (S.E.L. Mirski et al., Cancer Res., 47:2594, 1987). In the present study, we have used the polymerase chain reaction to verify that H69AR cells do not overexpress P-glycoprotein. Further, transport studies with radiolabeled daunomycin, VP-16, and vinblastine demonstrate that differences in net drug accumulation or efflux are not part of the resistance phenotype of H69AR cells. To determine if H69 and H69AR cells differ in their susceptibility to drug-induced DNA damage, DNA single-strand breaks (SSB) generated by VP-16 and Adriamycin were measured using the alkaline filter elution assay. Readily detectable SSB were produced in intact H69 cells by 5 microM VP-16, but 100 microM drug was required to cause similar damage in H69AR cells. H69AR cells were also resistant to SSB induction by Adriamycin. The formation of SSB by VP-16 was similarly reduced in isolated H69AR nuclei, indicating that resistance to this drug resides, at least in part, in the nucleus. No significant differences were observed in the rate or extent of repair of VP-16-induced DNA SSB in H69 and H69AR cells. The reduced susceptibility to drug-induced SSB may result from alterations in topoisomerase II, since less immunoreactive topoisomerase II was found in H69AR cells compared to H69 cells. However, changes in topoisomerase II cannot explain the resistance of H69AR cells to such drugs as the Vinca alkaloids and gramicidin D, indicating that multiple mechanisms contribute to drug resistance in this small cell lung cancer cell line.