We have recently reported the crystal structure of the accessory subunit of mitochondrial DNA polymerase, pol gammaB, and identified a region of the protein involved in DNA binding. The DNA employed in previous studies was presumed to be single-stranded, because it was generated by single-sided PCR. Further characterization of this DNA indicated that, due to a strand transfer event during synthesis by single-sided PCR, the DNA adopts a double-stranded hairpin conformation under native conditions. We used a series of double- and single-stranded oligonucleotides of different lengths to confirm that human pol gammaB prefers to bind double-stranded DNA longer than 40 bp with little apparent sequence specificity. Site-specific deletion mutagenesis identified clusters of basic residues in two surface loops required for DNA binding located on opposite sides of the symmetrical pol gammaB dimer. A heterodimer of pol gammaB that contains one mutant and one wild-type DNA binding region was shown to be unable to bind double-stranded DNA, suggesting that a single DNA molecule must contact both DNA binding sites in the pol gammaB dimer. The ability to bind double-stranded DNA is not essential for pol gammaB stimulation of pol gammaA activity in vitro, but may play a role in DNA replication or repair.