SET, the translocation breakpoint-encoded protein in acute undifferentiated leukemia (AUL), is a 39-kDa nuclear phosphoprotein and has an inhibitory activity for protein phosphatase 2A (PP2A). SET is fused to a putative oncoprotein, CAN/NUP214, in AUL and is thought to play a key role in leukemogenesis by its nuclear localization, protein-protein interactions and PP2A inhibitory activity. Here, we describe the isolation and characterization of a novel cDNA encoding a protein with 1542 amino-acid residues that specifically interacts in a yeast two-hybrid system as well as in human cells with SET. This new protein, which we name SEB (SET-binding protein), is identified as a 170-kDa protein by immunoprecipitation with a specific antibody and is localized predominantly in the nucleus. SEB1238--1434 is determined as a SET-binding region that specifically binds to SET182--223. SEB also has an oncoprotein Ski homologous region (amino acids 654--858), six PEST sequences and three sequential PPLPPPPP repeats at the C-terminus. SEB mRNA is expressed ubiquitously in all human adult tissues and cells examined. The SEB gene locus is assigned to the chromosome 18q21.1 that contains candidate tumor suppressor genes associated with deletions in cancer and leukemia. Although the function of SEB is not known, we propose that SEB plays a key role in the mechanism of SET-related leukemogenesis and tumorigenesis, perhaps by suppressing SET function or by regulating the transforming activity of Ski in the nucleus.