Flavopiridol, a synthetic flavone that inhibits tumor growth in vitro and in vivo, is a potent cyclin-dependent kinase (cdk) inhibitor presently in clinical trials. In the present study, the effect of 100-500 nM flavopiridol on a panel of non-small cell lung cancer cell lines was examined. All express a wild-type retinoblastoma susceptibility protein and lack p16INK4A, and only A549 cells are known to express wild-type p53. During 72 h of treatment, flavopiridol was shown to be cytotoxic to all seven cell lines, as measured by trypan blue exclusion, regardless of whether cells were actively cycling. In most cycling cells, cytotoxicity was preceded or accompanied by cell cycle arrest. Cell death resulted in the appearance of cells with a sub-G1 DNA content, suggestive of apoptosis, which was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and by demonstration of cleavage of caspase targets including poly(ADP-ribose) polymerase, p21Waf1, and p27Kip1. At doses at or below 500 nM, maximal cytotoxicity required 72 h of exposure. Although flavopiridol resulted in the accumulation of p53 in A549 cells, flavopiridol-mediated apoptosis was p53 independent because it occurred to the same degree in A549 cells in which p53 was targeted for degradation by HPV16E6 expression. The data indicate that flavopiridol has activity against non-small cell lung cancers in vitro and is worthy of continued clinical development in the treatment of this disease.