To characterize the enhanced repair synthesis of defined DNA lesions, oligodeoxyribonucleotides were synthesized and inserted into plasmid DNA. The inserted plasmid DNA was treated with cis-diamminedichloroplatinum(II) (cisplatin) and subjected to in vitro DNA repair assay with soluble extract from the rat liver cell line Ac2F. All cisplatin adducts tested stimulated DNA repair synthesis. Moreover, two cisplatin-resistant cell lines, Ac2F-CR4 and Ac2F-CR10, were established by stepwise exposure of Ac2F cells to this drug. The DNA repair synthesis was enhanced 3- to 4-fold in the extract from cisplatin-resistant Ac2F cells relative to that from Ac2F cells. Such repair synthesis was suppressed by the specific DNA polymerase inhibitor aphidicolin. The results of the present study suggested that the enhanced repair activity induced by a cisplatin adduct can be detected by in vitro DNA repair assay with soluble cell extract.