In order to define the most effective combination schedule of oxaliplatin (L-OHP) and 5-fluorouracil (5-FU), we investigated the in vitro interaction between these drugs in a panel of four human gastric adenocarcinoma cell lines (MKN-1, NUGC-3, NUGC-5 and AZ-521). Cytotoxic activity was determined by the WST-1 assay. Different schedules of the two drugs were compared and evaluated for synergism, additivity or antagonism with a quantitative method based on the median-effect principle of Chou and Talalay. Cell cycle perturbation and apoptosis were evaluated by flow cytometry. Simultaneous and sequential treatments of L-OHP followed by 5-FU exhibited synergistic effects in all four cell lines, whereas the reverse sequence yielded a clear antagonism. 5-FU exclusively arrested cells at the G0/G1 phase, and L-OHP at the G0/G1 and G2/M phases. Apoptosis was most prominent when cells were treated simultaneously or in a sequence of L-OHP followed by 5-FU, producing apoptosis in the majority of treated cells (55.5-61.5%). In contrast, the reverse sequence yielded only 20% induction of apoptosis, the rate being not significantly different from those induced by each drug singly. Moreover, this sequence dependence was further confirmed by the experiment which compared the total number of NUGC-3 cells 7 days after these combination schedules. These findings suggest that the interaction of 5-FU and L-OHP could be highly schedule dependent, with the most efficacious interaction observed in simultaneous combination and that 5-FU followed by L-OHP would not be recommended in clinical trials for patients with advanced gastric cancer.