An Mg(2+)-dependent deoxyribonuclease activity was highly purified from a 0.6 M NaCl extract of rat-liver nuclei. The template primer activity assay with a Klenow polymerase suggested that this nuclease plays a role in incision/excision of cisplatin-modified DNA strands to form single-strand gaps. In another experiment, rat-liver (Ac2F) cells were cultured in the presence of cisplatin. A 0.6 M NaCl extract was prepared from the cultured-cell nuclei and subjected to the activity blotting analysis [Seki et al. (1993) J. Chromatography 618, 147-166]. The nuclease activity of the extract was enhanced in response to cisplatin, but not in the presence of cycloheximide. These results imply that cisplatin-DNA lesions induce the Mg(2+)-dependent deoxyribonuclease activity in Ac2F cells to provide priming sites for the repair synthesis.