Anion‐exchange chromatography of partially purified human HL‐60 topoisomerase II resolves the known α (170 kDa) and β (180 kDa) isoenzymes at 150 mM NaCl and 230 mM NaCl, respectively. An additional topoisomerase II fraction was eluted by > 300 mM NaCl. It could be identified by Western blotting as a late‐eluting variant of topoisomerase IIα, which is functionally altered as compared to the early‐eluting form, having the following properties: a shift in the catalytic optimum to pH 9; increased stability in DNA complex formation; approximately 100‐fold resistance to orthovanadate; approximately 1000‐fold resistance to the cytostatic substances N‐4‐(9‐acridinylamino)‐3‐methoxyphenyl‐methanesulphonamide (amsacrine) and the podophyllotoxin etoposide (VP 16). 80% of the late‐eluting topoisomerase IIα could be captured by SDS on calf thymus DNA without further enhancement by drugs. In contrast, the early‐eluting topoisomerase IIα exhibits 10% complex formation with SDS alone, and an increase to 90% complex formation in the presence of drugs. A HL‐60 subline (HL‐60/R), approximately 1000‐fold resistant to etoposide and amsacrine, has equivalent proportions of topoisomerase IIα and topoisomerase IIβ and similar levels of both isoenzymes, as compared to the drug‐sensitive HL‐60/WT cells. However, determination of the cellular levels of the early‐eluting and late‐eluting forms of topoisomerase IIα revealed that the HL‐60/R cell line contains approximately 80% of the late‐eluting topoisomerase IIα, whereas the sensitive HL‐60/WT cell line contains only 15–20% of this form. The nuclear distribution of the two forms also differs. Sensitive HL‐60/WT cells show a diffuse nuclear distribution but in resistant cells the distribution is localized in the nucleoli. Apparently two functionally distinct subforms of topoisomerase IIα coexist in drug‐sensitive and drug‐resistant HL‐60 cells and changes in their relative levels affect the cellular sensitivity to topoisomerase‐II‐targeting drugs.