The Runt domain transcription factor, PEBP2/CBF, is a heterodimer composed of 2 subunits. The DNA-binding α subunit, or RUNX protein, interacts with a partner PEBP2β/CBFβ through the evolutionarily conserved Runt domain. Each of the genes encoding RUNX1 and PEBP2β/CBFβ is frequently involved in acute myeloid leukemia. The chimeric protein, CBFβ(PEBP2β)/SMMHC, is generated as a result of inversion of chromosome 16 in such a way to retain the heterodimerization domain of PEBP2β at the amino-terminal side fused to the C-terminal coiled-coil region of smooth muscle myosin heavy chain (SMMHC). Here we show that, in the chimeric protein, the second heterodimerization domain is created by the fusion junction, enabling the chimeric protein to interact with RUNX1 at far greater affinity than PEBP2β and inactivate the RUNX1/AML1 function. To explain why and how heterozygous CBFB/MYH11 can inactivate homozygous RUNX1 near to completion, we propose a new model for this chimeric protein that consists of a Y-shaped dimer with unpaired N-terminal halves followed by a coiled-coil for the C-terminal region. (Blood. 2004;103:3200-3207)