Polyomavirus enhancer binding protein, PEBP2 (PEA2), is a heterodimer of two distinct subunits, α and β, of which the former directly binds to DNA and the latter acts auxiliary to enhance the DNA binding. Recent cloning studies has revealed that the α subunit is homologous to the products of the Drosophila segmentation gene runt and the human AML1 gene, and that it functions as a major regulator for the T cell-specific gene expression. We have currently cloned cDNAs for the β subunit. The isolated cDNAs contain three isoforms that are presumed to arise from alternative RNA splicing and encode polypeptides consisting of 187, 182, and 155 amino acids, respectively. These polypeptides neither show any significant homology with known other proteins including the α subunit nor have any known DNA-binding and dimerization domains. Thus, PEBP2, as the complex of these subunits, is thought to constitute an entirely novel category of heteromeric transcriptional regulator together with the Runt and AML1 proteins. Gel retardation assays of the cDNA-encoded proteins produced in an in vitro translation system or in Escherichia coli demonstrated that the larger two β isoforms, but not the smallest one, can dimerize with the α subunit. Furthermore, this heterodimerization was shown to cause a marked increase in the intrinsic DNA binding affinity of the α subunit.