Listeria monocytogenes causes a pro-inflammatory response on adhesion to macrophages. Upregulation of inflammation genes involves the transcription factor NF-kappaB. Several components of L. monocytogenes, including lipoteichoic acid (LTA), phospholipases and listeriolysin O (LLO), have since been shown to mediate NF-kappaB activation. Here, we report that purified recombinant InlB, but not internalin (InlA), is a potent activator of NF-kappaB in the mouse macrophage-like cell line J774. Expression of InlB in Listeria innocua enhances its ability to activate NF-kappaB, while deletion of InlB from L. monocytogenes marginally decreases its effect on NF-kappaB, possibly because of the presence of NF-kappaB activators such as LTA and LLO. The effect correlates with the rapid degradation of IkappaBalpha, a sustained degradation of IkappaBbeta and increases in tumour necrosis factor alpha (TNF-alpha) and interleukin (IL) 6 production, two cytokines controlled by NF-kappaB. Using a series of anti-InlB monoclonal antibodies and domains of InlB, NF-kappaB activation was shown to be dependent upon the N-terminal 213-amino-acid leucine-rich repeat (LRR) domain of InlB, recently demonstrated to be responsible for InlB-mediated L. monocytogenes invasion and phosphoinositide-3 (PI-3) kinase activation. The effect of InlB was blocked by PI-3 kinase inhibitors, indicating the involvement of PI-3 kinase in this response. This report thus illustrates that InlB not only promotes invasion, but also contributes to the macrophage pro-inflammatory response.