Chemotherapy agents have been shown to induce the transcription factor nuclear factor-kappaB (NF-kappaB) and subsequent chemoresistance in fibrosarcomas and other cancers. The mechanism of NF-kappaB-mediated chemoresistance remains unclear, with a previous report suggesting that doxorubicin induces this response independent of the inhibitory kappaB kinases (IKK). Other studies have indicated that IKKbeta, but not IKKalpha, is required. Mouse embryo fibroblasts devoid of IKKalpha, IKKbeta, or both subunits (double knockout) were treated with doxorubicin. The absence of either IKKalpha or IKKbeta or both kinases resulted in impaired induction of NF-kappaB DNA-binding activity in response to doxorubicin. To provide a valid clinical correlate, HT1080 human fibrosarcoma cells were transfected with small interference RNA specific for IKKalpha or IKKbeta and then subsequently treated with doxorubicin. Knockdown of IKKalpha severely impaired the ability of doxorubicin to initiate NF-kappaB DNA-binding activity. However, a decrease in either IKKalpha or IKKbeta resulted in decreased phosphorylation of p65 in response to doxorubicin. The inhibition of doxorubicin-induced NF-kappaB activation by the knockdown of either catalytic subunit resulted in increased cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase and increased apoptosis when compared with doxorubicin alone. The results of this study validate current approaches aimed at NF-kappaB inhibition to improve clinical therapies. Moreover, we show that IKKalpha plays a critical role in NF-kappaB-mediated chemoresistance in response to doxorubicin and may serve as a potential target in combinational strategies to improve chemotherapeutic response.