Analyses of the interactions of rat polymerase β (rat pol β) with a double-stranded DNA have been performed using the quantitative fluorescence titration and fluorescence energy transfer techniques. The obtained results show that rat pol β binds to dsDNA oligomers with the site-size of the enzyme dsDNA complex n = 5 ± 1 base pairs. The small site-size of the complex is a consequence of engagement of only the 8-kDa domain in intrinsic interactions with the dsDNA. This conclusion is directly supported by the fluorescence energy transfer between the single tryptophan residue on the 31-kDa domain and fluorescence acceptor located on the DNA. The dsDNA oligomer is bound at a distance of at least 55 Å from the tryptophan, excluding the 31-kDa domain from any closed contact with the DNA, Moreover, in the complex with the dsDNA, the enzyme is bound in "open" conformational state, The intrinsic interactions are accompanied by a net release of about four to five ions. The net ion release is dominated by cations as a result of the exclusive engagement of the 8-kDa domain in interactions. Magnesium affects the net ion release through direct binding of Mg2+ cations to the protein, Surprisingly, binding of rat pol β to the dsDNA is characterized by strong positive cooperative interactions, a very different behavior from that previously observed for pol β complexes with the ssDNA and gapped DNAs. Contrary to intrinsic affinities, cooperative interactions are accompanied by a net uptake of about three to five ions. Anions have a large contribution to the net ion uptake, indicating that cooperative interactions characterize protein - protein interactions. The significance of these results for the pol β functioning in damaged-DNA recognition processes is discussed.