The ability to monitor transcription factor (TF) activation in the central nervous system (CNS) has the potential to provide novel information regarding the molecular mechanisms underlying a wide range of neurobiological processes. However, traditional biochemical assays limit the mapping of TF activity to select time points. In vivo bioluminescence imaging (BLI) has emerged as an attractive technology for visualizing internal molecular events in the same animal over time. Here, we evaluated the utility of BLI, in combination with virally-mediated delivery of reporter constructs to cardiovascular nuclei, for monitoring of TF activity in these discrete brain regions. Following viral gene transfer of NF-B-driven luciferase reporter to the subfornical organ (SFO), BLI enabled daily measurements of baseline TF activity in the same animal for one month. Importantly, systemic endotoxin, a stimulator of NF-B activity, induced dramatic and dose-dependent increases in NF-B-dependent bioluminescence in the SFO up to 30 days after gene transfer. Co-treatment with a dominant-negative IB mutant significantly prevented endotoxin-dependent NF-B activation, confirming the specificity of the bioluminescence signal. NF-B-dependent luminescence signals were also stable and inducible one month following delivery of luciferase reporter construct to the paraventricular nucleus or rostral ventrolateral medulla. Lastly, using targeted adenoviral delivery of an AP-1 responsive luciferase reporter, we showed similar baseline and endotoxin-induced AP-1 activity in these same brain regions as with NF-B reporters. These results demonstrate that BLI, in combination with viral-mediated gene transfer, is a powerful method for longitudinal monitoring and quantification of TF activity in targeted CNS nuclei in vivo.