Each of the two human genes encoding the alpha and beta subunits of a heterodimeric transcription factor, PEBP2, has been found at the breakpoints of two characteristic chromosome translocations associated with acute myeloid leukemia, suggesting that they are candidate proto-oncogenes. Polyclonal antibodies against the alpha and beta subunits of PEBP2 were raised in rabbits and hamsters. Immunofluorescence labeling of NIH 3T3 cells transfected with PEBP2 alpha and -beta cDNAs revealed that the full-size alpha A1 and alpha B1 proteins, the products of two related but distinct genes, are located in the nucleus, while the beta subunit is localized to the cytoplasm. Deletion analysis demonstrated that there are two regions in alpha A1 responsible for nuclear accumulation of the protein: one mapped in the region between amino acids 221 and 513, and the other mapped in the Runt domain (amino acids 94 to 221) harboring the DNA-binding and the heterodimerizing activities. When the full-size alpha A1 and beta proteins are coexpressed in a single cell, the former is present in the nucleus and the latter still remains in the cytoplasm. However, the N- or C-terminally truncated alpha A1 proteins devoid of the region upstream or downstream of the Runt domain colocalized with the beta protein in the nucleus. In these cases, the beta protein appeared to be translocated into the nucleus passively by binding to alpha A1. The chimeric protein containing the beta protein at the N-terminal region generated as a result of the inversion of chromosome 16 colocalized with alpha A1 to the nucleus more readily than the normal beta protein. The implications of these results in relation to leukemogenesis are discussed.