The polyomavirus enhancer binding protein 2alpha (PEBP2alpha) is a DNA binding transcriptional regulatory protein that binds conserved sites in the polyomavirus enhancer, mammalian type C retroviral enhancers and T-cell receptor gene enhancers. Binding of PEBP2alpha and homologous proteins to the consensus DNA sequence TGPyGGTPy is mediated through a protein domain known as the runt domain. Although recent NMR studies of DNA-bound forms of the runt domain have shown an immunoglobulin-like (Ig) fold, the identification of residues of the protein that are involved in DNA binding has been obscured by the low solubility of the runt domain. Constructs of the mouse PEBP2alphaA1 gene were generated with N- and C-terminal extensions beyond the runt homology region. The construct containing residues Asp90 to Lys225 of the sequence (PEBP2alpha90-225) yielded soluble protein. The residues that participate in DNA binding were determined by comparing the NMR spectra of free and DNA-bound PEBP2alpha90-225. Analysis of the changes in the NMR spectra of the two forms of the protein by chemical shift deviation mapping allowed the unambiguous determination of the regions that are responsible for specific DNA recognition by PEBP2alpha. Five regions in PEBP2alpha90-225 that are localized at one end of the beta-barrel were found to interact with DNA, similar to the DNA binding interactions of other Ig fold proteins.