Synergistic cytotoxicity between cisplatin and the nucleoside analog gemcitabine was observed in a panel of cisplatin-sensitive (2008, A2780) and -resistant (2008/C13*5.25, A2780/CP70) human ovarian cell lines. Previous studies have suggested a role for DNA repair in the mechanism of synergy between the two drugs. We therefore further investigated the hypothesis that the synergistic cytotoxicity between gemcitabine and cisplatin in these cell lines may be caused by gemcitabine-mediated inhibition of cisplatin intrastrand adduct (IA) and interstand cross-link (ICL) repair. The effect of gemcitabine on the accumulation and repair of cisplatin IA and ICL in each cell line was then measured directly using gene-specific quantitative polymerase chain reaction and denaturation/renaturation techniques, respectively. Pretreatment of 2008 cells with 1 microM gemcitabine for 2 h before exposure to cisplatin for 7 h enhanced the accumulation of cisplatin IA and ICL by 50 and 40%, respectively (P < 0.05), above that induced by cisplatin alone. To investigate the possibility that the increased accumulation of cisplatin lesions was caused by inhibition of removal of cisplatin damage, 2008 cells were incubated with 200 microM cisplatin for 5 h in the presence and absence of gemcitabine and then a further 8 h in the absence of cisplatin. Only 57% IA were removed in the combination treated cells compared with 74% in cisplatin control cells. Similarly, repair of cisplatin ICL was inhibited in the gemcitabine-treated cells compared with the cells treated with cisplatin only (60 versus 72%). These findings demonstrate a direct inhibitory effect of gemcitabine on the repair of cisplatin IA and ICL and suggest a mechanistic basis for the cytotoxic synergy between the two drugs.