An optimal scheduling of hyperthermia and cis-diammine-dichloroplatinum(II) (cisplatin) may increase the therapeutic gain of the combination of these two modalities. In this study, intracellular platinum accumulation, total platinum binding to DNA, and DNA interstrand crosslinks (ISC) were assayed to investigate the molecular mechanisms responsible for the effect of sequencing hyperthermia on the thermal enhancement of cisplatin-induced cytotoxicity in mouse leukemia L1210 cells. Simultaneous treatment with heat (41.5 degrees C, 60 min) and cisplatin produced maximal cell killing with a 4-fold decrease in the 50% growth-inhibitory concentration (IC50) of the platinum complex. Super-additive cell killing was also shown when cells were exposed to heat before cisplatin treatment, whereas no thermal enhancement in cisplatin-mediated cytotoxicity was observed in cells given heat after exposure to cisplatin. These results correlated with the degree of formation of ISC observed in cells following various treatments. A 2- to 3-fold increase in ISC formation was observed in cells given heat before or during cisplatin exposure, whereas heat after cisplatin treatment did not alter either the formation or the reversal of ISC as compared with cisplatin alone. The increased ISC formation was associated with an increase in intracellular platinum accumulation and total platinum binding to DNA in cells given heat before or during cisplatin exposure. These data, showing that hyperthermia potentiates cisplatin cytotoxicity by increasing drug uptake and the formation of DNA adducts without inhibiting the repair of DNA lesions, demonstrate the potential utility of sequencing hyperthermia combined with cisplatin as a clinical anticancer therapy.