A wide range of human disorders involves inappropriate regulation of NF-κB, including cancers and numerous inflammatory conditions. Toward our goal to define mechanisms through which NF-κB leads to the development of disease, we have developed transgenic mice that express luciferase under the control of NF-κB, enabling real-time in vivo imaging of NF-κB activity in intact animals. We show that in the absence of extrinsic stimulation, strong luminescence is evident in lymph nodes in the neck region, thymus, and Peyer’s patches. Treating mice with TNF-α, IL-1α, or LPS increased the luminescence in a tissue-specific manner, with the strongest activity observed in skin, lungs, spleen, Peyer’s patches, and the wall of the small intestine. Liver, kidney, heart, muscle, and adipose tissue displayed less intense activities. Also, exposure of skin to a low dose of UV radiation increased luminescence in the exposed areas. Furthermore, induction of chronic inflammation resembling rheumatoid arthritis produced strong NF-κB activity in the affected joints, as revealed by in vivo imaging. Thus, we have developed a versatile model for monitoring NF-κB activation in vivo.