A new dihydrofolate reductase inhibitor, edatrexate (EDX), and the microtubule polymerization promotor, taxol (TXL) or taxotere (TXT), each have significant therapeutic activity against human breast cancer in clinical trials. Since they also have distinctly different mechanisms of actions and have mainly non-overlapping toxicities, they may be effective in combination in the treatment of this disorder. Schedule-dependent interactions between these taxanes and EDX against human breast adenocarcinoma cells (SK-Br-3) were quantitatively assessed in vitro to determine whether these interactions are synergistic or antagonistic. SK-Br-3 cells were grown as a monolayer in 96-well microplates. The dose-effect relationships of the drugs, singly and in combination, in inhibiting the growth over a 7-day period were determined by the SRB protein staining assays. Cell cultures were exposed to drug as a 3-h pulse at either 0-3 h or 24-27 h. Synergism or antagonism at different concentrations and at different effect levels were assessed with the median effect principle and the combination index-isobologram method using computer software. These methods were selected because they take into account both the potencies and the shape of the dose-effect curves. Exposure of cells to an equimolar combination of EDX + TXL (0-3 h) resulted in synergism at high effect levels. Pretreatment of cells with EDX (0-3 h) followed by TXL (24-27 h) showed even greater synergism in inhibiting cell growth. Moderate antagonism was observed with the reverse schedule. EDX + TXT (0-3 h) was additive, but pretreatment with EDX (0-3 hr) followed by TXT (24-27 h) showed synergism. However, the reverse order showed antagonism. Studies on another breast tumor cell line, ZR-57-1, also showed the schedule of EDX (0-3 h) + TXT or TXL (24-27 h) to be more synergistic than, the other two schedules examined. These results show potent schedule-dependent synergism of the combinations of TXL or TXT with EDX, and should form a rationale for designing clinical protocols utilizing these agents particularly for the treatment of breast cancer patients.