Eucaryotic topoisomerase II is an essential nuclear enzyme involved in processes such as chromosome condensation, chromatid separation, and in the relief of torsional stress that occurs during DNA transcription and replication. In cells from vertebrate species, there are two forms of the enzyme, designated alpha and beta. Human topoisomerase IIalpha (TOP2A) is encoded by the TOP2A gene on chromosome 17q21-22, and human topoisomerase IIbeta (TOP2B) is encoded by the TOP2B gene on chromosome 3p24. The protein products of these two genes are important cellular targets of several drugs widely used in the treatment of many human cancers, and a variety of mutations in TOP2A have been associated with the development of drug resistance. In the present study, we have defined the intron-exon structures of TOP2A and TOP2B. TOP2A is approx. 30kb whereas TOP2B is at least 49kb. TOP2A and TOP2B contain 35 and 36 exons, respectively, and both genes contain a high proportion of class 0 introns. Alignment of the amino-acid sequences of the two proteins indicates that the intron-exon organization of the two genes is highly conserved, except for the regions encoding the extreme NH2 and COOH termini of the proteins. These findings suggest strongly that the vertebrate isoforms evolved by duplication of an ancestral gene. Mutations in TOP2A associated with drug resistance show clustering in exons 12, 13, 19-21 and 34-35. Knowledge of the genomic organization of TOP2A and TOP2B will be useful for detection of mutations in clinical samples from patients with drug-resistant malignant disease.