The combination of oxaliplatin (LOHP)-5-fluorouracil (FU)-folinic acid (FA) has provided high response rates in pretreated patients with advanced colorectal cancer that is resistant to FU-FA. However, the choice of the optimal schedule between LOHP, FU, and FA remains open. The purpose of the present study was to compare, at equivalent drug area under the curve, different schedules for the LOHP-FU +/- FA combinations on four human colorectal cancer cell lines. FU +/- FA was tested as a 2-h short exposure ("bolus"), a 118-h continuous exposure ("infusion"), or a 22-h mixed exposure ("De Gramont protocol"). LOHP was administered for 2 h before, during, or after FU +/- FA exposure. Isobologram analyses revealed that LOHP associated with FU +/- FA resulted in synergistic cytotoxic effects whatever the tested schedules (in > or = 75% of cases). For the FU-LOHP combination, cytotoxicity was significantly different according to the FU exposure type (short > mixed > continuous) and was independent of the LOHP position. In contrast, for the FU-FA-LOHP combination, neither the FU exposure type nor the LOHP position significantly influenced cytotoxicity. The presence of FA significantly enhanced the cytotoxicity of FU-LOHP (P < 0.001); this potentiation was independent of the FU exposure type and was significantly influenced by the LOHP position (LOHP after FU-FA > LOHP during FU-FA > LOHP before FU-FA). In conclusion, in contrast with the recognized superiority of continuous FU exposure over short exposure when the drug is given alone, the FU-LOHP combination is more cytotoxic when FU is given as a short exposure. This suggests the potential interest of such a schedule in the clinical setting.