We investigated the cytotoxic effect of different sequential combinations of paclitaxel (Taxol, TX), an antimicrotuble agent, and methotrexate (MTX), a potent inhibitor of dihydrofolate reductase, on a human bladder cancer cell line (T24). The results obtained with a colony-forming assay 5 days after 24 hr of exposure to TX alone showed a strong decline in colony growth up to a concentration of 6 nM. There was a plateau phase at TX concentrations ranging from 6 to 10 nM. The IC50 values found with the different dosing sequences and 24-hr exposure to the drugs were 0.24 microM for MTX alone, 3.49 nM for TX alone, 3.84 nM for TX followed by MTX, 3.75 nM for TX and MTX simultaneously, and 2.10 nM for MTX followed by TX. The cytotoxic effect due to drug interactions in the different sequential combinations was evaluated by an algebraic method. All the combination sequences were found to be synergistic (combination index [CI] < 1) at low TX concentrations. An antagonistic effect (CI > 1) was obtained at higher TX concentrations. Our data demonstrate that the T24 cell line is sensitive to TX and that the cytotoxic effect is enhanced to different degrees depending on the treatment sequence when TX is combined with MTX. These findings could have implications in the design of appropriate administration schedules of these two drugs for the treatment of bladder cancer.