DNA topoisomerase II alpha is a nuclear enzyme essential for DNA metabolism and cell cycle progression. Previous studies have shown that human tumor cell lines can express more than one topoisomerase II alpha isoform through alternative splicing. A 160-kDa isoform of topoisomerase II alpha has been described in several cell lines selected for resistance to inhibitors of DNA topoisomerase, but its physiological function has not been defined. In the present study, we have identified two major (160 and 140 kDa) and two minor (150 and 145 kDa) isoforms of topoisomerase II alpha in drug-sensitive human leukemic CEM cells, all of which have lost C-terminal regions that produce epitopes recognized by specific antibodies. Reverse transcription-polymerase chain reaction and molecular cloning identified four alternatively spliced transcripts of topoisomerase II alpha from CEM cells. Furthermore, nucleotide sequencing indicated that the 160-kDa isoform is encoded by two transcripts derived from alternative splicing at a different C-terminal site and that the other two transcripts likely code for the 150-kDa isoform. Although the full-length topoisomerase II alpha resided in the cell nucleus, all altered isoforms, except the 160 kDa that was located in both cytoplasmic and nuclear extracts in about equal amount, were shown to be present predominantly in the cytosol. In contrast to the observations of other groups, we have not found an association of the topoisomerase II alpha isoforms with drug resistance. Rather, our results suggest that expression of topoisomerase II alpha isoforms is cell type specific or might be associated with the neoplastic phenotype of the cells. Thus, although T-lineage tumor cell lines examined (CEM, Jurkat, and H9) displayed altered topoisomerase II alpha isoforms, normal T cells expressed only a full-length copy of the gene. Together, these results suggest that expression of altered topoisomerase II alpha isoforms is not limited to drug resistance, but might be a feature of neoplastic cells.