NF-κB is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Although the transactivation ability of NF-κB has been extensively studied in vitro, limited information is available on the spatial and temporal transactivation pattern in vivo. To investigate the kinetics and cellular localization of NF-κB-induced transcription, we created a transgenic mouse expressing the enhanced GFP (EGFP) under the transcriptional control of NF-κB cis elements (cis-NF-κB EGFP). A gene-targeting approach was used to insert a single copy of a NF-κB-dependent EGFP reporter gene 5′ of the X-linked hypoxanthine phosphoribosyltransferase locus in mouse embryonic stem cells. Embryonic fibroblasts, hepatic stellate cells, splenocytes, and dendritic cells isolated from cis-NF-κB EGFP mice demonstrated a strong induction of EGFP in response to LPS, anti-CD3, or TNF-α that was blocked by the NF-κB inhibitors BAY 11-0782 and NEMO-binding peptide. Chromatin immunoprecipitation analysis demonstrated RelA binding to the cis-NF-κB EGFP promoter. Adenoviral delivery of NF-κB-inducing kinase strongly induced EGFP expression in the liver of cis-NF-κB EGFP mice. Similarly, mice injected with anti-CD3 or LPS showed increased EGFP expression in mononuclear cells, lymph node, spleen, and liver as measured by flow cytometry and/or fluorescence microscopy. Using whole organ imaging, LPS selectively induced EGFP expression in the duodenum and proximal jejunum, but not in the ileum and colon. Confocal analysis indicated EGFP expression was primarily found in lamina propria mononuclear cells. In summary, the cis-NF-κB EGFP mouse will serve as a valuable tool to address multiple questions regarding the cell-specific and real-time activation of NF-κB during normal and diseased states.