Abnormal expression of the nuclear-associated enzyme DNA topoisomerase II (topoisomerase II) has been implicated in the in vitro phenotype of radiation hypersensitive ataxia-telangiectasia (A-T) cells and in modifying sensitivity of eukaryotic cells to topoisomerase II-inhibitor drugs [e.g., the DNA intercalator amsacrine (mAMSA)]. To study such relationships, various SV40- and Epstein-Barr Virus-transformed human cell lines derived from normal, A-T, or UV-sensitive xeroderma pigmentosum donors have been assayed for their sensitivity to mAMSA together with direct and indirect measurements of topoisomerase II expression. We report on the identification of an SV40-transformed A-T fibroblast cell line with abnormally high levels of topoisomerase II in nuclear protein extracts as determined by immunoblotting, measurement of kinetoplast DNA decatenation activity, and mAMSA-dependent DNA-protein cross-linking activity in a filter binding assay. Using a flow cytometric assay for the analysis of reactivity of nuclei with a polyclonal antitopoisomerase II antibody, overproduction was found to occur in all phases of the cell cycle. High levels of topoisomerase II were associated with hypersensitivity (5-10-fold) to mAMSA-induced cell cycle delay, cell kill, and DNA strand breakage (assayed under protein-denaturing conditions). Xeroderma pigmentosum (group A) cells demonstrated normal responses to mAMSA. The results provide evidence that cellular potential for the generation of topoisomerase II-dependent DNA damage is a major factor in governing the sensitivity to mAMSA. Furthermore, underexpression of topoisomerase II does not appear to be a primary factor in describing the in vitro A-T phenotype. The findings also relate to how changes in chromatin structure and function may either reflect or dictate the expression of topoisomerase II in human cells.