The nucleoporin 98 kDa (NUP98) gene has been reported to be fused to 18 different partner genes in various hematological malignancies with 11p15 aberrations. The most frequently observed fusion partners of NUP98 belong to the homeobox family of transcription factors, whereas the non-HOX NUP98 fusion partners comprise a heterogeneous group of genes that are associated with a wide range of biological functions. Cytogenetic analysis of an adult de novo acute myeloid leukemia (AML-M5a) revealed a t(3;11)(p24;p15) indicating fusion of NUP98 with a novel partner gene. Fluorescence in situ hybridization (FISH) analysis with the NUP98-specific clone 1173K1 showed a split signal, suggesting that NUP98 was indeed disrupted. Selection of possible NUP98 partner genes was performed by computer-aided analysis of the 3p24 region using the University of California Santa Cruz genome browser. Out of the genes located at 3p24, TOP2B was selected as a fusion partner candidate gene. Dual-color fusion gene-specific FISH and RT-PCR analyses verified that NUP98 was indeed fused to TOP2B. In addition to the reciprocal NUP98-TOP2B and TOP2B-NUP98 in-frame fusion transcripts, an alternatively spliced out-of-frame TOP2B-NUP98 transcript that resulted in a premature stop codon was detected. Analysis of the genomic breakpoints revealed typical signs of non-homologous end joining resulting from error-prone DNA repair. TOP2B encodes a type II topoisomerase, which is involved in DNA transcription, replication, recombination, and mitosis. Type II DNA topoisomerases exist as homodimers, with each subunit consisting of three functional domains: an N-terminal ATPase domain, a central DNA breakage-rejoining domain, which contains a nucleotide-binding motif and the catalytic tyrosine, and a relatively poorly conserved C-terminal domain. The C-termini of the two TOP2 isoforms seem to be important for subcellular localization and functional bipartite nuclear localization signal (NLS) sequences as well as nuclear export signals (NES) are located in these domains. The NUP98-TOP2B fusion transcript fuses the N-terminal FG repeat and GLEBs motifs of NUP98 with the C-terminal domain of TOP2B, thereby retaining the functional NLS but eliminating the NES. Consequently, the putative reciprocal TOP2B-NUP98 chimeric protein retains the ATPase, the DNA breakage-rejoining, and the NES domains of TOP2B that are fused to the ribonucleoprotein-binding and the NLS domains of NUP98. The shorter out-of-frame TOP2Bexon24-NUP98exon14 fusion transcript might encode a truncated TOP2B isoform that consists of the ATPase, the DNA breakage-rejoining, and NES domains, which are fused to 18 fusion partner-unrelated amino acids. All proteins encoded by non-HOX NUP98 fusion partners described to date contain regions with a significant probability to adopt a coiled-coil conformation, and protein analysis with the COILS 2.2 and the MULTICOIL programs revealed this remarkable feature also in the C-terminal region of TOP2B. Intriguingly, this is only the second description of a chromosomal rearrangement that involves a topoisomerase and both TOP1 and TOP2B are fused to NUP98. This suggests that the choice of partner genes for NUP98 is not random, and that NUP98-TOP fusions may represent a distinct group, similar to the NUP98-HOX fusions.