The transcription factor NF-kappaB is frequently activated in cancer, and is therefore a valuable target for cancer therapy. Decoy oligodeoxynucleotides (ODNs) inhibit NF-kappaB by preventing its binding to the promoter region of target genes. Few studies have used NF-kappaB-targeting with ODNs in cancer. Using a hairpin NF-kappaB-decoy ODN we found that it induced growth inhibition and cell death in NF-kappaB-dependent tumour cell lines. The ODN colocalized with the p50 subunit of NF-kappaB in cells and directly interacted with it in nuclear extracts. In TNFalpha-treated cells the ODN and the p50 subunit were found in the cytoplasm suggesting that the complex did not translocate to the nucleus. Transcriptional activity of NF-kappaB was efficiently inhibited by the ODN, whereas a scrambled ODN was without effect on transcription. Thus, ODN-mediated inhibition of NF-kappaB can efficiently promote cell death in cancer cells providing a potentially powerful approach to tumour growth inhibition.