The t(12;13)(p13;q12) is a rare, recurrent translocation reported in a range of hematological malignancies. We have analyzed the molecular basis of this lesion in three patients with acute myeloid leukemia (AML), two of whom were known to have chromosome 12 breakpoints within the ETV6 gene. Fluorescence in situ hybridization (FISH) with ETV6 cosmids indicated that this gene was also disrupted in the third patient, while the normal ETV6 allele was retained. 3′ rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) from bone marrow mRNA of this individual identified a novel sequence fused to ETV6 that was homologous to a region just upstream of the mouse CDX2 homeobox gene, the human homologue of which has previously been mapped to chromosome 13q12. PCR primers designed to amplify an ETV6-CDX2 fusion identified two major transcripts from this patient. First, a direct in-frame fusion between exon 2 of ETV6 and exon 2 of CDX2, and second, a transcript that had an additional sequence of unknown origin spliced between these same exons. Surprisingly, apparently normal CDX2 transcripts, usually expressed only in intestinal epithelium, were also detectable in cDNA from this patient. Neither normal nor fusion CDX2 mRNA was detectable in the two other patients with a t(12;13), indicating that this translocation is heterogeneous at the molecular level. Reverse transcription-PCR analysis showed that CDX2 mRNA, but not ETV6-CDX2 mRNA, was strongly expressed in 1 of 10 patients with chronic myeloid leukemia in transformation, suggesting that deregulation of this gene may be more widespread in leukemia. CDX2 is known to regulate class I homeobox genes and its expression in hematopoietic cells may critically alter the balance between differentiation and proliferation.