Exposure of lung cancer cells to gemcitabine (2',2'-difluorodeoxycytidine) arrests cells in S phase and induces secondary apoptotic cell death. Gemcitabine treatment decreased the expression of IkappaB-alpha protein and, concomitantly, increased the activity of nuclear factor-kappaB (NF-kappaB) transcription factor, a known inhibitor of the apoptotic response. This increase was accompanied by a similar increment in the expression of inhibitor of apoptosis-1 (IAP-1) protein and mRNA, a caspase inhibitor responsive to NF-kappaB. These changes were important to the final destiny of the cells, since overexpression of a dominant negative version of IkappaB-alpha, which suppresses NF-kappaB activation, blocks the increase of IAP-1 protein and potentiates the action of gemcitabine. Additionally, overexpression of IAP-1 protein in A549 cells expressing the IkappaB-alpha mutant restored the initial sensitivity to gemcitabine and demonstrated that this protein was responsible for the inhibitory effect of NF-kappaB. These results support the notion of IAP-1 as an important antiapoptotic protein mediating sensitivity to deoxynucleotides analogs in non-small-cell lung cancer cells.