We showed previously that a sequential treatment with doxorubicin (4 hr) followed by paclitaxel (24 hr) (Dox=Pacl) induces a synergistic cytotoxic effect in the BRC-230 breast cancer cell line and in human primary breast cancer cultures. The validity of this experimental finding was confirmed in a clinical phase I/II study on advanced breast cancer patients. To improve the cytotoxic effect obtained by the Dox=Pacl sequence, we analyzed the effect of adding gemcitabine (Gem) to the Dox=Pacl sequence in a preclinical study. Our study was performed on BRC-230 and MCF-7 cell lines, and cytotoxic activity was evaluated by the sulforhodamine B assay and the type of drug interaction by Drewinko's test. When Gem (0.01 g/ml for 24 hr) was given immediately or 24 hr after Dox=Pacl, an antagonistic cytotoxic effect was observed. Conversely, a synergistic effect was found when Gem was given 48 hr after Dox=Pacl. From results of flow cytometric analysis, the synergistic effect was attributed to cell cycle perturbation. Cells were arrested in G 2 -M (95% in treated vs. 21% in control samples) 24 hr after Dox=Pacl treatment. The block progressively recovered thereafter, and after a further 24 hr, at the time of Gem treatment, the cells progressed into the G 1 -S phase boundary (the cell cycle phase susceptible to the cytocidal effect of the drug). Our findings suggest that the interactions of Dox, Pacl and Gem are highly schedule-and time-dependent and should be taken into consideration in the planning of clinical protocols.