Flavopiridol is a potent inhibitor of cyclin-dependent kinases (cdks). In a large proportion of solid tumor cell lines, the initial response to flavopiridol is cell cycle arrest. NCI-H661 non-small cell lung cancer cells are representative of a subset of more sensitive cell lines in which apoptosis is observed during the first 24 h of drug exposure. Analysis of the apoptotic population indicates that cells in S-phase are preferentially dying. In addition, cells are sensitized to flavopiridol following recruitment to S-phase, whether accomplished by synchronization or by treatment with noncytotoxic concentrations of chemotherapy agents that impose an S-phase delay. Combinations of gemcitabine or cisplatin, followed by flavopiridol at concentrations that correlate with cdk inhibition, produce sequence-dependent cytotoxic synergy. A survey of paired cell lines, including WI38 diploid fibroblasts or normal human bronchial epithelial cells, along with their SV40-transformed counterparts, demonstrates that treatment with flavopiridol during S-phase is selectively cytotoxic to transformed cells. These data suggest that treatment during S-phase may maximize responses to flavopiridol and that the administration of flavopiridol after chemotherapy agents that cause S-phase accumulation may be an efficacious antitumor strategy.