DNA-binding non-histone proteins (NHP) from rat liver have been prepared by utilizing their affinity toward phosphocellulose. The specifity of the DNA binding sites was investigated by the reassociation kinetics of the protein-bound DNA. Isolated protein-bound DNA (350 nucleotides) reassociated with a a-fold increase in the content of middle repetitive sequences as found in the whole rat genome. Kinetic analysis of the bound DNA reassociation suggests that half of the total repetitive sequences was concentrated by the NHP interactions. Saturation reassociation of isolated middle repetitive DNA or the whole rat genome, of various fragment lengths, with an excess of bound DNA indicates 5% or less of the rat genome is complementary with the driver DNA. This finding confirms the previous observations that a subset of the repetitive sequences is concentrated by NHP-DNA interactions (Sevall, J. S., Cockburn, A., Savage, M., and Bonner, J. (1975) Biochemistry 14,782-789). Increasing the DNA fragment length to 3400 nucleotides enhanced the amount of NHP-retained DNA. Reassociation of sheared bound 3400 nucleotide-DNA resulted in two kinetic components, the slower component being nonrepetitive and the fast component being repetitive. The repetitive sequences displayed reassociation kinetics identical with those of the NHPbound 350-nucleotide fragments. The NHP-bound nonrepetitive component was 10% of the whole single copy sequence complexity. Rat liver polysomal RNA-driven hybridization of the protein-bound nonrepetitive component had a 4to 5-fold increase in complementary DNA fragments when compared with the whole rat genome nonrepetitive sequences even though this represents only 10% of the total genome.