Nuclear factor (NF)-kappaB is frequently over-expressed in non-small cell lung cancer (NSCLC), but the exact role of this observation remains unclear. In this regard, activation of the transcription factor may govern distinct steps of NSCLC progression, such as carcinogenesis, angiogenesis, and metastasis. In these studies we attempted to dissect the effects of two proteins of the NF-kappaB pathway (p65/RelA and IkappaBetaalpha) on experimental metastasis of murine NSCLC, using a novel approach of bioluminescent detection of NF-kappaB activation in tumor cells. Stable integration of a NF-kappaBeta reporter confirmed high basal activation of the transcription factor in mouse NSCLC cells in vitro and during experimental metastasis to the lungs, like human NSCLC. In the mouse model of NSCLC metastasis, NF-kappaBeta-dependent luciferase expression served as a reliable indicator of tumor cell delivery to the lungs, establishment of metastatic tumors, and lung tumor burden. In vitro transient p65/RelA and IkappaBetaalpha gene transfer to mouse NSCLC cells resulted, respectively, in significant NF-kappaB activation and inhibition, without affecting cell growth. However, p65/RelA overexpression in NSCLC cells drastically reduced in vivo metastasis to the lungs, while overexpression of IkappaBetaalpha had no effect. In conclusion, using bioluminescent detection of NF-kappaB activation in mouse lug adenocarcinoma cells, we found a negative impact of p65/RelA on NSCLC metastasis.