The high-mobility-group (HMG) chromosomal protein wheat HMGa was purified to homogeneity and tested for its binding characteristics to double-stranded DNA. Wheat HMGa was able to bind to P268, an A/T-rich fragment derived from the pea plastocyanin gene promoter, producing a small mobility shift in gel retardation assays where the bound complex was sensitive to addition of proteinase K but resistant to heat treatment of the protein, consistent with the identity of wheat HMGa as a putative HMG-I/Y protein. Gel retardation assays and southwestern hybridization analysis revealed that wheat HMGa could selectively interact with the DNA polynucleotides poly(dA).poly(dT), poly(dAdT).poly(dAdT), and poly(dG).poly(dC), but not with poly(dGdC).poly(dGdC). Surface plasmon resonance analysis determined the kinetic and affinity constants of sensor chip-immobilized wheat HMGa for double-stranded DNA 10-mers, revealing a good affinity of the protein for various dinucleotide combinations, except that of alternating GC sequence. Thus contrary to prior reports of a selectivity of wheat HMGa for A/T-rich DNA, the protein appears to be able to interact with sequences containing guanine and cytosine residues as well, except where G/C residues alternate directly in the primary sequence.