Camptothecin-resistant DC3F Chinese hamster lung fibroblast cell lines were obtained after mutagenic treatment with ethylmethanesulfonate and subsequent exposure to 1 microM camptothecin (CPT). The most resistant cell line, which was obtained after exposure to CPT for 10 days, was designated DC3F/C-10. Comparison of 50% inhibitory concentration values after 8-h CPT treatments showed that DC3F/C-10 cells were 134-fold resistant to CPT. Resistance was associated with marked reduction of CPT-induced DNA single-strand breaks and DNA-protein cross-links. This reduction was not due to reduced amounts of immunoreactive DNA topoisomerase I protein, although nuclear extracts from DC3F/C-10 cells had less enzyme catalytic activity than those from DC3F cells. Also, fast protein liquid chromatography-purified DNA topoisomerase I from DC3F/C-10 had lower specific catalytic activity than that from DC3F cells. DNA topoisomerase I from DC3F/C-10 was resistant to inhibition of catalytic activity and induction of DNA cleavage by CPT. These results suggest that CPT resistance in DC3F/C-10 cells is due to qualitative alteration of DNA topoisomerase I.