Fractionation of nuclear extracts from a multi-drug-resistant subclone of the human promyelocytic subline HL-60 by anion-exchange chromatography and chromatofocusing resolves at least two different subtypes of topoisomerase II, which are not identical to the known alpha- and beta-forms of the enzyme because both forms are contained in each subtype. The two subtypes are present in about equal proportions and differ remarkably with respect to the optimum of reaction and sensitivity to m-amsacrine and orthovanadate. Both subtypes are highly insensitive to etoposide inhibition in vitro.