Flavopiridol, the first potent cyclin-dependent kinase inhibitor to undergo clinical trials as an antineoplastic agent in the United States, has attracted considerable attention because of its unique cellular targets and its ability to kill noncycling tumor cells in vitro. To better understand how flavopiridol might be used clinically, the present study used colony-forming assays to examine the cytotoxicity resulting from combining flavopiridol with eight other antineoplastic agents in four different administration schedules in A549 human non-small cell lung carcinoma cells in vitro. Cytotoxic synergy, as assessed by the median effect method, resulted when flavopiridol was combined with seven of the eight tested antineoplastic agents but was highly dependent upon administration schedule. Cisplatin was the only agent that resulted in sequence-independent synergy when combined with flavopiridol. For paclitaxel, cytarabine, topotecan, doxorubicin, and etoposide, synergy was more pronounced when the agents were administered before flavopiridol rather than concomitant with or following flavopiridol. Examination suggested that this sequence dependence reflected arrest of cells in G1 and G2 phases of the cell cycle during and for 24 h following flavopiridol treatment. Interestingly, 48-72 h after flavopiridol removal, the fraction of surviving cells in S phase increased 2-3-fold relative to untreated controls. Consistent with these results, administration of flavopiridol for 24 h followed 3 days later by exposure to an S phase-active agent (cytarabine or 5-fluorouracil) resulted in a highly synergistic interaction. These results highlight the importance of administration schedule when combining flavopiridol with other agents and provide a starting point for examining the effect of flavopiridol in drug combinations in vivo.