Fludarabine (9-beta-arabinofuranosyl-2-fluoroadenine-5'-monophosphate) is clinically active against chronic lymphocytic leukemia and low-grade lymphomas. We reported previously that fludarabine nucleoside synergistically enhanced cisplatin (CDDP)-induced cytotoxicity in vitro, and that the synergism was concomitant with inhibition of removal of cellular CDDP-induced DNA interstrand cross-links, which are presumably repaired by homologous recombinational repair. To extend our work, we investigated whether fludarabine inhibits nucleotide excision repair (NER) of CDDP-induced DNA intrastrand adducts. The effect of fludarabine on NER was determined using a cell-free system in which a plasmid containing the DNA adducts served as the substrate for repair enzymes in whole-cell extracts from repair-competent cells. To prevent the cell-bound high mobility group box-containing proteins from interfering with repair, cell extracts were depleted with high mobility group box proteins by immunoprecipitation prior to the assay. Repair synthesis, measured by the incorporation of [(32)P]dATP or [(32)P]dCTP, was inhibited by 50% at 26 or 43 microM fludarabine triphosphate, respectively; the effect was dose dependent and may have resulted from the termination of repair-patch elongation. These results were consistent with those from pulse-chase experiments demonstrating the conversion of nicked circular plasmid to the closed circular form by cell extracts filling the repair gaps. When proliferating cell nuclear antigen-depleted cell extracts were used and aphidicolin was added in the repair assay to arrest NER at the incision/excision stage, 100 microM fludarabine triphosphate inhibited about 55% of the conversion of nicked plasmids from the closed circular damaged plasmid substrate; the inhibition was dose dependent. We conclude that fludarabine triphosphate inhibited NER at the steps of incision and repair synthesis. These results suggest that fludarabine may serve as a potential repair modulator to improve the antitumor efficacies of combination regimens containing agents that induce NER.