We found that nervonic acid (NA, 15cis-tetracosenoic acid) which is a cis-configurated unsaturated long-chain fatty acid and lithocholic acid (LCA, hydroxy-5β-cholan-24-oic acid) which is a bile acid are selective inhibitors of mammalian DNA polymerase β (pol β) and DNA topoisomerase II (topo II). Here, we report the molecular interaction of NA and LCA with pol β or topo II. On 1H - 15N HMQC NMR analysis of pol β with NA or LCA, the 8 kDa domain of pol β bound to NA or LCA as a 1 : 1 complex with a dissociation constant (KD) of 2.64 or 1.56 µM, respectively. The NA-binding region was comprised mainly of four amino acid residues (Leu11, Lys35, His51 and Thr79) of pol β on the NA-interaction interface. Similarly, the LCA-binding region consisted of three amino acid residues (Lys60, Leu77 and Thr79). Based on a three-dimensional structural analysis and comparison with the spatial positioning of specific amino acids binding to NA and LCA on pol β, we obtained supplementary information allowing us to build a structural model of topo II using geometrical and evolutionary trace methods. The four amino acid residues were Thr596, His735, Leu741 and Lys983 for topo II, corresponding to Thr79, His51, Leu11 and Lys35 for pol β and the three amino acid residues were Lys720, Leu760 and Thr791 for topo II, corresponding to Lys60, Leu77 and Thr79 for pol β. These results suggested that the NA and LCA-binding domains of pol β and topo II are three-dimensionally very similar.